Loss of pancreas upon activated Wnt signaling is concomitant with emergence of gastrointestinal identity

RESEARCH ARTICLE Loss of Pancreas upon Activated Wnt SignalingIs Concomitant with Emergenceof GastrointestinalIdentity Jose Luis Muñoz-Bravo 1,2☯ , Alvaro Flores-Martı ´nez 1,2☯ , Griselda Herrero-Martin 1,2 , Sapna Puri 3 , Makoto Mark Taketo 4 , Anabel Rojas 5,6 , Matthias Hebrok 3 , David A. Cano 1,2 * 1Unidad de Gestio ´n Clı ´nica de Endocrinologı ´a y Nutricio ´n, Hospital Universitario Virgen del Rocı ´o, Sevilla, Spain, 2Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocı ´o/Consejo Superior de Investigaciones Cientı ´ficas/Universidad de Sevilla, Sevilla, Spain, 3Diabetes Center, Department of Medicine, University of California San Francisco, San Francisco, United States of America, 4Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 5Centro Andaluz de Biologı ´a Molecular y Medicina Regenerativa (CABIMER), Sevilla, Spain, 6Centro de Investigacio ´n Biome ´dica en Red de Diabetes y Enfermedades Metabo ´licas Asociadas (CIBERDEM), Madrid, Spain ☯These authors contributed equally to this work. *[email protected] Abstract Organ formation is achieved through the complex interplay between signaling pathways and transcriptional cascades. The canonical Wnt signaling pathway plays multiple roles during embryonic development including patterning, proliferation and differentiation in distinct tissues. Previous studies have established the importance of this pathway at multiple stages of pancreas formation as well as in postnatal organ function and homeostasis. In mice, gain-of-function experiments have demonstrated that activation of the canonical Wnt pathway results in pancreatic hypoplasia, a phenomenon whose underlying mechanisms remains to be elucidated. Here, we show that ectopic activation of epithelial canonical Wnt signaling causes aberrant induction of gastric and intestinal markers both in the pancreatic epithelium and mesenchyme, leading to the development of gut-like features. Furthermore, we provide evidence that β-catenin-induced impairment of pancreas formation depends on Hedgehog signaling. Together, our data emphasize the developmental plasticity of pancreatic progenitors and further underscore the key role of precise regulation of signaling pathways to maintain appropriate organ boundaries. Introduction Organ formation during embryonicdevelopment is controlled by a relatively small number of signalingpathways. These pathways, however, need to be precisely regulated in a spatio-tempo- ral manner for the correct development of tissues and organs. Improper activation (or inhibition) of these pathways can result in severe impairment of cell specificationand differentiation. The pancreas derivesfrom the endodermalgermlayer duringgastrulation.In mice, the pancreas PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 1 / 22 a11111 OPEN ACCESS Citation: Muñoz-Bravo JL, Flores-Martı ´nez A, Herrero-Martin G, Puri S, Taketo MM, Rojas A, et al. (2016) Loss of Pancreas upon Activated Wnt Signaling Is Concomitant with Emergence of Gastrointestinal Identity. PLoS ONE 11(10): e0164714. doi:10.1371/journal.pone.0164714 Editor: Gregory M. Kelly, Western University, CANADA Received: March 2, 2016 Accepted: September 29, 2016 Published: October 13, 2016 Copyright: ©2016 Muñoz-Bravo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and Supporting Information files. Funding: This work was supported by Nicola ´s Monardes program of Andalusian Ministry of Health (www.juntadeandalucia.es/ fundacionprogresoysalud/es), Grant number: C- 0015-2014 (DAC); Andalusian Ministry of Science and Innovation (www.juntadeandalucia.es/ innovacioncienciayempresa), Grant number: CTS- 7478 (DAC); Ministry of Economy and Competitiveness, Spain (www.mineco.gob.es), Grant number: SAF2011-26805 (DAC); Ministry of develops from two independentprimordia(dorsal and ventral) of the foregut epithelium around 8.5–9 days of gestation (E8.5–9). The pancreatic epithelium expands and branches into the surrounding mesenchyme by E11.5. This early pancreatic epithelium contains multipotent pancreatic progenitor cells that give rise to all adult pancreatic cell types. Around E13.5, two distinct cellulardomains are formedin the pancreatic epithelium. The epithelial "tips" of the branching epithelium becomeacinar progenitor cells while the "trunk"domain contains cells that will give rise to endocrineand ductal cells. This period is known as the "secondarytransition", in which extensive endocrineand exocrinecell differentiationtakes place. After the secondarytransition, the differentiated pancreatic cells undergo furthergrowth and differentiationwith endocrine cells organizing into islets of Langerhans. The development of the mammalian pancreas has been intensively studied with the goal of generating ß-cells in vitro for transplantation therapy, significantly advancing our understandingof the underlying molecularmechanisms duringthe last severaldecades(see[1–4] for recent reviewson the subject). The pancreas is of great medicalrelevance due to the devastating diseasesassociatedwith failure of this organ, including diabetes,pancreatitis and pancreatic cancer. Much of the molecular knowledgegained in pancreas development has also contributed to shed light on the mechanisms underlying adult organ homeostasisand metaplasia. Thus, it has becomeincreasingly evident that many of the embryonic signalingpathways involved in pancreas development such as Hedgehog (Hh), Notch and Wnt signalingalso play crucialroles in pancreas regeneration and cancer in the adult organism [5–8]. However, less is known about the link betweenthese embryonicsignaling pathways and developmental anomalies of the pancreas in humans. The study of these rare conditions in humans has revealedmutations in genes encoding master transcriptionfactors that regulate pancreas embryonic formation in mice (reviewed in [1,9]) but the role of signalingpathways remains largely unexplored. Previous studies have established the importanceof the canonical Wnt signalingpathway in embryonic pancreas formation as well as in postnatal organ functionand homeostasis [10–13]. Thesestudies have revealeda complex role of the canonical Wnt pathway on pancreas development fulfillingdifferent functions at distinctstages of embryonic pancreas development. This is best illustrated by gain-of-functionstudies that have illuminatedthe importanceof the timing of canonical Wnt activationduring pancreas development.Precociousactivation of β-cate- nin in the early stages of pancreas formation in mice (betweenembryonicday E10.5-E12.5) results in pancreas hypoplasia, while activation at a later time point duringembryogenesis causes a marked increase in pancreas organ size [11,14]. Similarly, overexpression of Wnt ligands in the early pancreatic domain causes severepancreatic hypoplasia [13,15]. While these studies indicate an inhibitory role of the canonical Wnt signalingpathway during early stages of pancreas formation, the underlying mechanisms remain to be elucidated. Also, while canonical Wnt signalingpathway seems to be important for the specificationand patterning of several endoderm-derivedorgans such as intestine [16] and lung [17], whetherthis pathway plays a role in the initial specificationand morphogenesis of the pancreas remains unclear. In the present study, we show that activation of β-catenin in the prepancreatic endoderm impairs pancreas formation. Moreover, we demonstrate that ectopic β-catenin activation can redirect the developmental fate of pancreatic progenitors towards a gastrointestinal identity, a process mediated by Hedgehog signaling. Materials and Methods Mice All procedures involving experimentalanimals were performedin accordance with European and local animal welfare laws, guidelinesand policies.All experimentalprotocols were Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 2 / 22 Economy and Competitiveness, Spain (www. mineco.gob.es), predoctoral fellowship number: BES-2009-012084 (JLMB); Ministry of Economy and Competitiveness, Spain (www.mineco.gob.es), Ramon y Cajal Program number: RYC-2013-14533 (AR); Instituto de Salud Carlos III, Spain (www. isciii.es) cofunded by Fondos FEDER, Grant Number: PI14/00804 (AR); and National Institute of Health, US (www.nih.gov) Grant Number: DK105831 (MH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. approved by the IBiS-Virgen del RocioEthics Committee and by the Committeeon Animal Research at the University of California,San Francisco (permitnumber AN088473-01C). Animals were euthanizedby cervicaldislocation. Pdx1-Cre [18], Rfx6-Cre [19] and Ctnnb1 tm1Mmt/+ [20] mice carryingthe floxed exon 3 allele of β-catenin have been previously described.Noon of the day when vaginal plugs are detected is treated as E0.5 day post coitum. Histological analyses Tissue processing,histologicaland immunohistochemicalanalyses were performedas previously described[21]. Immunoperoxidase staining was performedwith the ABC Elite and DAB kits (Vector). For Alcian Blue staining, deparaffinizedslides were incubatedin a solution of 10g/l 8GX Alcian blue pH 2.5 for 30 min, washed and counterstained with nuclear fast red (NFR). For Periodic acid-Schiffstaining (PAS), deparaffinizedslides were incubated in 0.5% periodicacid solution for 5 min at room temperature, rinsed,placed in Schiffreagent (Sigma- Aldrich) for 15 min and counterstained with hematoxylin. For Gomori's one-step trichrome staining,deparaffinizedslides were pretreated with Bouin'ssolution (Sigma-Aldrich)for 1 hour at 56°C, placed in Weigert's iron hematoxylin for 10 min, stained with Gomori trichrome stain (Sigma-Aldrich)for 15 min and differentiatedin 0.5% aceticacid for 2 minutes. Fluorescent and bright-fieldimages were captured using a BX-61 microscope(Olympus) or LTC SP2 confocal microscope(Leica).Primaryantibodiesusedare listed in S1 Table. All photomicrographs shown are representative of at least three independent samples of the indicated genotype. To quantify Cpa1-positivearea at E13.5, the entire pancreatic tissue was sectionedin 5 μm thick sectionsand everyfifth sectionwas immunostained for Cpa1. The total pancreatic area, identifiedby morphologyand localization,were measured using ImageJ software. For double Cpa1 and E-cadherinimmunofluorescence,Cpa1-positivearea was expressed as a ratio of total epithelial E-cadherin area to visualize epithelial area. Pancreas explants E10.5 pancreatic rudiments and guts were dissectedtogetherand cultured in the air-liquid interface for 3 days on Millicell (Millipore) inserts as previously reported [22]. Briefly, guts were harvestedin Hank’s balanced salts solution (HBSS) and cultured in 24-well plates with 350 μl of RPMI mediumsupplied with10% fetal bovineserum,containing 100 U/ml penicillin, 100 μg/ml streptomycin and 2mM of L-glutamine. Cyclopamine (C4116 Sigma-Aldrich)was dissolved in DMSO (stock solution 10 mM) and added to the culture at a final concentration of 10 μM as previously described[23]. Medium was changed everyother day. Whole Mount Immunostaining Cultured explants were permeabilizedand cleared as previously reported[24] with the following modifications.Cultured explants were fixed for 30 min at 4°C in 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS). Primary and secondaryantibodieswere incubated at 4°C for 24 and 48 hours respectivelywith gentle rocking.z-stack confocalimages were taken every1 μm using a LTC SP2 confocal microscope(Leica).Area quantification was performed with ImageJ (NIH). Quantitative real-time PCR For RNA isolation, dorsal pancreatic buds at different stages were quickly dissectedin cold PBS and placed into 10 volumes (w/v) of RNAlater (Qiagen) at 4°C over-night. Total RNA was Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 3 / 22 isolated using the RNeasy Kit (Qiagen) following manufacturer instructions.DNAse I treatment (Sigma)was usedprior to retrotranscriptasereactionto ensure elimination of all genomic DNA. cDNA was synthesized using Omniscript reverse transcriptase (Qiagen). Real-time quantitative PCR was performedwith SYBR Green PCR Master Mix (Applied Biosystems) using a 7900HT real-time PCR system (Applied Biosystems). Relative quantification of RNA levels was calculatedusing ΔΔCt method.Cyclophilin A (peptidylprolyl isomeraseA—Mouse GenomeInformatics) was selectedfrom a panel of 6 reference genes as the most stable. Results are expressed as fold relative to levels in control pancreata (value of 1). Primers were selected and validated for gut and pancreatic tissue in accordance to MIQE guidelines[25]. Primer sequencesare listed in S2 Table. Microarray Analysis Pancreata were dissectedfrom E13.5 embryosand stored in RNAlater (Qiagen) until genotypedwas performed.Once genotyped,3 pancreata from Pdx1-Cre; Ctnnb1 tm1Mmt/+ and control embryos were pooledand total RNA was isolated using the RNeasy Kit (Qiagen) following manufacturer instructions.Expression analysis was performedin three independent poolsof both Pdx1-Cre; Ctnnb1 tm1Mmt/+ and control pancreata using AffymetrixMouse Gene 1.0 ST Array (Affymetrix,Santa Clara, CA) following manufacturer's instructions at CABIMER GenomicCore Facility. Statistical analysis of the microarray data was done by LIMMA (Linear Models for Microarray Analysis) using R software. This analysis applies a t-test empirically adjusted by a Bayes test. Microarray data were depositedin Gene Expression Omnibus database (GEO; accession number:GSE82342). Statistical analysis Significancewas determinedusing two-tailedStudent’s t-test, and one-way ANOVA (post-hoc Tukey HSD test). p<0.05 was considered significant.Data are presented as mean ± s.e.m. Results Activation of Wnt/ β-catenin induces loss of pancreatic identity To evaluate the effectof canonical Wnt pathway activation on pancreas formation, we crossed mice carryinga floxed exon 3 activating allele of β-catenin (Ctnnb1 tm1Mmt/+ ) [20] with mice expressing Cre recombinase undercontrol of the pancreatic and duodenalhomeobox1 (Pdx1) promoter (Pdx1-Cre mice)[18].Confirmingprevious studies [11], we found that newborn mutant Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice displayed a dramatic reductionof pancreatic tissue in both the splenic and gastric lobes (S1 Fig). The pancreatic remnant was clearly distinguishable from adjacent stomach and duodenum (S1 Fig). Histological and immunofluorescence analysis revealedthe presence of epithelial cysts and abundant stroma in the pancreatic remnant (S1 Fig). Scarcepancreatic tissue with normal appearance could also be observed(S1 Fig). However, in these cells, β-catenin localizationwas restrictedto the membrane with no apparent nuclear or cytoplasmicaccumulation (S1 Fig) likely due to inefficientexcision of the Ctnnb1 tm1Mmt/+ transgene by the Cre recombinase. To determinethe specificperiodin which β-catenin activation impaired embryonic pancreas formation, we analyzed Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata at various developmental stages. The first phenotypes were observedbetween E12.5-E13.5,with the pancreatic epithelium of Pdx1-Cre; Ctnnb1 tm1Mmt/ embryosdisplaying abnormal lumen dilations (Fig 1A and 1B). BetweenE13.5 and E15.5, a dramatic morphogeneticremodelingoccurs in the pancreatic epithelium that results in the formation of different progenitor domains, with proacinar Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 4 / 22 Fig 1. Loss of pancreatic cell identity in Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreas. Hematoxylin/eosin staining of E13.5 pancreatic sections showing reduced branching and multiple dilations in the pancreatic epithelium of Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata (A) compared to control pancreata (B). Boxed areas in A and B are shown in higher magnification in A’ and B’, respectively. The enzyme Carboxipeptidase A1 (Cpa1, red) is located in the acinar progenitor cell population found at the tips of the E13.5 branching epithelium stained for the epithelial maker E-cadherin (green) in control mice (C). Cpa1-positive cells (arrowheads) are reduced in E13.5 Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreatic epithelium (D). The pancreatic ductal epithelium in E13.5 Pdx1-Cre; Ctnnb1 tm1Mmt/+ embryos display loss of apical marker Muc-1 (green) Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 5 / 22 progenitor cells located at the tips of the branching epithelium and ductal-endocrineprecur- sors located along the epithelial trunk [26]. At E13.5, the control pancreas formed a branched epithelial (marked by E-Cadherinexpression) tree with tips positivefor CarboxypeptidaseA1 (Cpa1) (Fig 1C). In contrast, the Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreatic epithelium did not branch properly with a decreasein Cpa1-positivetips and abnormally expandedlumens (Fig 1D and 1G). The dilated epithelial areas were associatedwith loss of apical ductal markers such as Mucin-1 [27] (Fig 1E and 1F), although localizationof basal and basolateral ductal markers laminin and E-cadherinwas not affected (Fig 1E and 1F). By E15.5, the disorganization of the pancreatic epithelium and the lack of normal pancreatic tissue in Pdx1-Cre; Ctnnb1 tm1Mmt/+ embryoswere clearly apparent (S2 Fig). The epithelial dysmorphogenesisof Pdx1-Cre; Ctnnb1 tm1Mmt/+ embryonic pancreata was associated with defectivecell differentiation.Immunofluorescenceand quantitative RT-PCR analyses of embryonicpancreata revealed a significant decreasein the expression of transcriptionfactors essential for pancreas formation including Pdx1 (Fig 1H, 1I and 1N), Nkx2.2(Fig 1J, 1K and 1N), Nkx6.1 (Fig 1L, 1M and 1N), Ptf1a and Ngn3 (S2 Fig), Hhex and Sox9 (Fig 1N). Cells with normal levels of these pancreatic transcription factors were also found in Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata but, similar to what was observedin newbornpancreata, no nuclear β-catenin localization was observed(Fig 1I). To furthercharacterizethe molecularchanges induced by β-catenin activation,we performed whole transcriptome analysis on Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata. Microarray analysis confirmeda marked downregulation of pancreatic genes in Pdx1-Cre; Ctnnb1 tm1Mmt/+ E13.5 embryonic pancreata (Table 1). Consistent with the prominent nuclear accumulation of β-catenin, an increase in the expression of canonical Wnt signalingtarget genes was observed in Pdx1-Cre; Ctnnb1 tm1Mmt/+ embryonic pancreata indicating hyperactivation of the canonical Wnt signalingpathway (Fig 1O). Thus, ectopic β-catenin activation in the developing mouse pancreas severely impairs pancreatic epithelial tubulogenesisand cell differentiation. Canonical Wnt signaling activation leads to activation of gastric and intestinal gene expression The cystic structurespresent in newbornPdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata were lined by a single-layered cuboidalepithelium surrounded by dense mesenchyme (S1 Fig), features that are reminiscent of gut-likestructures.To definethe nature of the cysticstructures,we examined a number of gastric and intestinal markers in newbornPdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata. Alcian blue and periodicacid Schiff(PAS) staining revealed the presence of cells abnormally producing mucin in the pancreatic epithelium (Fig 2A–2D). In agreement with these observations,cells expressing gastric-specificmucin Muc5ac (Fig 2E and 2F) and cells expressing intestine-specificmucin Muc2 (Fig 2G and 2H) were found interspersed within the pancreatic epithelium of Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice. To further characterize the cystic structuresof Pdx1-Cre Ctnnb1 tm1Mmt/+ pancreata, we studied embryonicgastric and intestinal markers. Sox2 and Cdx2 are transcription factors essential for the formation of stomach and (arrowheads) but normal basal (laminin, blue) and basolateral marker (E-cadherin, green) in dilated areas (F) compared to control pancreata (E). Insets show higher magnification pictures. (G) Quantification of Cpa1-positive area. Marked reduction in Pdx-1 expression in Pdx1-Cre Ctnnb1 tm1Mmt/+ pancreatic epithelium (I) at E13.5 compared to control pancreata (H). Note that in Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreatic epithelium, Pdx-1 expression is dramatically reduced in cells displaying nuclear β-catenin accumulation while cells with only membranous βcatenin localization express significant Pdx-1 levels (I’ and I’’). Decreased expression of the embryonic pancreatic transcription factors NKX2.2 (J, K) and NKX6.1 (L, M) in E13.5 Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata. (N) Quantitative RT-PCR analysis of the expression of pancreatic transcription factors in E13.5 and E15.5 embryonic pancreas. (O) Quantitative RT-PCR analysis of the expression of canonical Wnt signaling target genes at E13.5 and E15.5 embryonic pancreas. Results in N and O are expressed as fold relative to levels in control pancreata (n = 3–6 embryos from each genotype). Data are presented as mean ±SEM; *p<0.05 (Student’s t-test). Scale bars 100 μm, (in A for A-B; in E for E-H; in I for I-J). doi:10.1371/journal.pone.0164714.g001 Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 6 / 22 intestine, respectively [28,29]. Duringembryonicdevelopment, Sox2 and Cdx2 are exclusively expressed in the foregut and intestinal lineages,respectively, thus permittinga clear distinction betweenstomach and intestine [30]. In newborncontrol pancreata, neither Sox2 nor Cdx2was expressed in the pancreatic epithelium as assessed by immunohistochemicalanalysis (Fig 2M and 2N). However, a strong Sox2 signal was observedin the epithelial compartment of cysts in Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata (Fig 2K and 2L). Interestingly, cells positive for the intestinal transcription factor Cdx2 were also found within the pancreatic epithelial cysts of Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice (Fig 2I and 2J). Colocalizationexperiments with Sox2 and Cdx2 revealedthat cells of Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata coexpress both markers (Fig 2O). To confirmthe originof the epithelial cysts of Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata, we performedgenetic lineage tracingexperimentsusing Pdx1-Cre to irreversibly activate the expression of the reporterR26R (ß-galactosidase).X-gal staining of newbornPdx1-Cre; R26R; Ctnnb1 tm1Mmt/+ pancreata revealed that the epithelial cells lining the cysts were positive for βgalactosidase(S1 Fig), a result consistent with a pancreatic origin.Immunohistochemistry analysis on X-gal-stainedsectionsrevealed that the Pdx1-derived cells expressed gut markers such as Sox2 (Fig 2P). Consistent with the immunohistochemicaldata, microarray analysis revealeda prominent increase in stomach progenitor genes such as Sox2,Pitx1 and Barx1 (Table 2 and Fig 2Q) and intestinal progenitor genes such as Cdx2, Lgr5 and FoxL (Table 2 and Fig 2Q) indicating aberrant activation of the gastrointestinal geneticprogram during early embryonicstages.Summarily, these results show that β-catenin activation in the pancreatic primordium results in loss of pancreatic identity with pancreatic cells adopting the morphological and molecularfeatures of gut-like cells. Table 1. Representative list of selected pancreatic genes downregulated in in Pdx1-Cre; Ctnnb1 tm1Mmt/+ E13.5 embryonic pancreata. Gene Fold change p-value Neurog3 -24.49 1.68E-07 Pdx1 -17.83 2.67E-09 Neurod1 -15.87 1.81E-07 Cpa1 -14.51 2.74E-08 Slc2a2 -10.06 5.49E-06 Nr5a2 -9.65 2.27E-08 Nkx2-2 -8.93 1.16E-08 Muc1 -7.86 8.50E-07 Onecut1 -7.72 1.36E-07 Iapp -6.64 8.52E-04 Bhlha15 -6.45 3.40E-04 Pax6 -6.11 7.48E-07 Nkx6-1 -5.72 1.59E-07 Rbpjl -5.01 1.99E-04 Ptf1a -4.93 2.68E-05 Hnf4a -4.89 1.55E-06 Glis3 -3.60 7.70E-07 Rfx6 -3.28 2.33E-05 Gata4 -2.77 1.26E-04 Myt1 -2.71 4.40E-04 Hhex -2.52 8.68E-05 Hnf1a -2.49 3.69E-05 doi:10.1371/journal.pone.0164714.t001 Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 7 / 22 Fig 2. β-catenin stabilization induces activation of gastrointestinal genes. Staining for PAS (A, B) and Alcian Blue (C, D) in Pdx1-Cre; Ctnnb1 tm1Mmt/+ and control newborn pancreata. Immunohistochemical staining for the gastric-specific mucin Muc5ac (E, F), intestine-specific mucin Muc2 (G, H) in control and mutant Pdx1-Cre; Ctnnb1 tm1Mmt/+ newborn pancreata. Arrowheads mark cells lining the epithelium positive for the gastrointestinal markers. Cdx2 accumulation in pancreatic cysts of newborn Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice in low (I) and high (J) magnification pictures. Cdx2 is not detected in newborn control pancreata (M). Sox2 accumulation in pancreatic cysts of newborn Pdx1-Cre; Ctnnb1 tm1Mmt/+ Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 8 / 22 Elevated canonical Wnt signaling causes a dramatic reorganization of the surrounding mesenchyme One of the most distinctivefeatures of Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata in newbornmice was the dramatic increase in mesenchymal tissue (S1 Fig). Trichrome staining revealed a thick mesenchyme layer surroundingthe epithelium of the cysticstructures in newbornPdx1-Cre; Ctnnb1 tm1Mmt/+ mice in contrast to the scarce mesenchymal tissue present in control mice (Fig 3A and 3B). To furthercharacterizethis expanded mesenchymal tissue, immunofluorescence analysis was performedwith antibodies against laminin and smooth muscle actin (SMA). In control pancreas, SMA was found only in big bloodvessels while laminin was detectedsurrounding all the epithelial structures(Fig 3C and 3E). Interestingly, in Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata, the cystic structures were surrounded by a basement membrane and a thick layer of mesenchyme tissue positivefor SMA (Fig 3D and 3F). A bilayer of SMA- positivecells with intercalated Tuj1-positive cells (indicative of the presence of neural cells), features characteristic of gut mesenchyme, was observedin some big pancreatic cysts in Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice (Fig 3F). This dramatic expansion of condensedmesenchyme seemedto be due to non-cell autonomous effects,secondaryto β-catenin activation in the epithelial compartment, as suggestedby the lack of nuclear β-catenin in mesenchymal cells of Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata (S1 Fig). Indeed, X-gal staining of Pdx1-Cre; R26R; Ctnnb1 tm1Mmt/+ pancreata revealed that cells positivefor β-galactosidasewere found only in the epithelial compartment (S1 Fig), rulingout an epithelial-mesenchymal transition phenomenon. Analysis of embryonicstages revealed expansion of SMA-positive mesenchymal cells as early as E13.5, concordant with the formation of dilated tubular epithelial structures (Fig 3G and 3H). At this stage, a significant increase in the proliferation of both mesenchymal and epithelial cells of embryonic Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata was noted (Fig 3I–3L). mice in low (K) and high (L) magnification pictures. No expression of Sox2 is detected in control pancreata (N). The boxed areas in I and K are shown at higher magnification in J and L, respectively. Arrowheads in J and L indicate the areas shown in the insets at higher magnification. (O) Coexpression of Sox2 and Cdx2 in epithelial cells of pancreatic cysts in newborn Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice. The boxed area in O is shown at higher magnification in the inset. (P) Coexpression of βgalactosidase and Sox2 gastric marker in epithelial cells (arrowheads) of pancreatic cysts in newborn Pdx1-Cre; R26R; Ctnnb1 tm1Mmt/+ mice. (Q) Analysis of the expression level of transcription factors specific of intestine (Cdx2) and stomach (Sox2 and Pitx1) showed a significant increase in expression in the pancreas of E15.5 Pdx1-Cre; Ctnnb1 tm1Mmt/+ embryos. Results are expressed as fold relative to levels in control pancreata (n = 6 embryos from each genotype). Data are presented as mean ±SEM; ** p<0.01 *** p<0.001 (Student’s t-test). st, stomach; d, duodenum. Scale bars, 100 μm. doi:10.1371/journal.pone.0164714.g002 Table 2. Representative list of selected intestinal and gastric genes upregulated in in Pdx1-Cre; Ctnnb1 tm1Mmt/+ E13.5 embryonic pancreata. Gene Fold change p-value Normal expression domain Lgr5 21.75 3.54E-09 Intestinal stem cells Foxf2 16.06 4.03E-07 Gut mesenchyme Foxf1a 11.63 1.18E-08 Gut mesenchyme Gata5 3.35 3.96E-06 Heart and gut FoxL1 3.31 3.47E-06 Gut mesenchyme Myocd 3.26 2.25E-06 Gut mesenchyme Acta2 2.83 1.67E-06 Smooth muscle Nkx2-3 2.12 1.72E-04 Gastrointestinal mesenchyme Pitx1 1.99 5.21E-04 Stomach Cdx2 1.97 3.32E-05 Intestine Sox2 1.61 3.29E-03 Stomach Barx1 2.39 7.74E-05 Stomach mesenchyme doi:10.1371/journal.pone.0164714.t002 Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 9 / 22 expression of transcription factors known to be required for pancreas formation. Also, epithelial branching morphogenesisand differentiation of the exocrineand endocrinelineages is markedly compromised. Concomitant with the downregulation of key pancreatic transcription factors, ectopic expression of gastric and intestinal genes is observed.At birth, pancreata of Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice were comprised of cystic structureswith features typical of stomach/intestine, including cuboidalepithelium, accumulation of mucins and a layer of SMA in the underlyingmesenchyme. Wnt signalingplays a key role in intestinal specificationand patterning [16]. It has been suggestedthat intestinal differentiationis the default program for the developinggut endoderm and that inhibition of canonical Wnt signalingis necessaryfor the formation of other foregutderivedorgans [33]. Forced activation of canonical Wnt signalingin developing endoderm[16, 39], lung [40] and stomach [41] induces intestinal differentiation.Our results demonstrate that activation β-catenin in pancreatic progenitors also induces ectopic expression of intestinal genes such as the transcription factor Cdx2 or the intestine-specificmucin Muc2. Cdx2 is a transcription factor whoseexpression is very specificto the intestine and that plays a critical role in intestinal development [28,30]. Expression of Cdx2 in foregut endoderminduces intestinal metaplasia [42]. Interestingly, induction of intestinal fate by ectopic Wnt signalingin endodermseems to be mediatedby Cdx2 [16]. In concordance with the pivotal role of Cdx2 in the inductionof an intestinal geneticprogram, increased expression of Cdx2 was observedas early as E13.5 in Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata. In addition to intestinal genes, activation of β-catenin resulted in the inductionof gastric markers in the pancreatic domain. In particular, we observedectopic expression of transcription factors that are essential for stomach development such as Sox2, Pitx1 and Barx1.Previous studies have shown that these markers are specificfor the anterior part of the primitive gut [29, 31]. Barx1 is particularlyinteresting since its expression is restrictedto the mesenchyme and exerts a non-cell autonomous effect on the overlying gut endoderm to regulate stomach development [41]. The inductionof stomach genes upon β-catenin activation in embryonicpancreata seems to be in apparent contradictionwith the above hypothesis that Wnt/β-catenin signalingpromotes intestinal fate. One possibleexplanation to reconcilethese observations comes from the proposed model for canonical Wnt signalingfunctionin stomach development [33,43]. Wnt signalingis activein the prospectivestomach endodermearly in development, betweenE9.5 and E14.5 [43]. However, canonical Wnt signalingactivity needs to be attenuated later to allowstomach differentiation.The inhibitionof canonical Wnt signalingin stomach is achievedby the transcription factor Barx1 whoseexpression is restrictedto the stomach mesenchyme and that induces the secretionof Wnt antagonists such as SFRPs [41]. Thus, although attenuation of canonical Wnt signalingis necessaryfor late stomach differentiation, a transient burst of canonical Wnt signalingactivity might be necessaryfor early embryonicstomach development. We propose that activation of β-catenin in the pancreatic epithelium induces a gastric genetic program including expression of the mesenchymal transcription factor Barx1. Even thoughincreasedexpression of Barx1 might induce secretionof Wnt antagonists, forced, irreversibleβ-catenin activation in the epithelium of Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata prevents a full transformation of the pancreas into stomach and consequently, inducingexpression of intestinal markers. In concordance with this notion, the mixed gastric/intestinal phenotype observedin pancreata with activated β-catenin is reminiscent of the stomach phenotypesof Barx1-deficientmice as well as stomach of mice with activation of β-catenin in the prospectivestomach endoderm[41]. The mix of gastric and intestinal markers inducedby β-catenin activation in pancreatic cells togetherwith the delay in the full activation of these markers (strong accumulation as assessed by immunohistochemistryis observedonly during late embryonic stages of pancreas Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 16 / 22 formation) raises the question of whetherthis can be considered a true fate conversion event. An alternative possibilityis that activation of β-catenin blocks pancreas differentiation leaving the pancreatic cellsin the default program for the developing gut endodermresulting in the formation of aberrant gut progenitor cells that express both gastric and intestinal markers. In agreement with this notion, colocalizationexperiments revealed the presence of cells that simultaneously express Cdx2 and Sox2 in Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata. Hh signalingis active and necessaryfor the embryonic development of the gut but excluded from the pancreas [35,44]. Our experimentsperformedin vitro with pancreatic rudiment cultures demonstrate that Hh signaling at least in part mediates the pancreatic phenotypes due to β-catenin activation.Inhibition of the Hh pathway by the Smoothenedinhibitor cyclopamine partiallyrescuespancreatic formation in Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreatic rudiments.To this regard, the Hedgehogpathway plays an important role in the development of the gastrointestinal mucosa, including proliferation of mesenchymal progenitors and differentiation of the smooth muscle lineage [36], in agreement with the increasedproliferation we observedin the mesenchymal compartment of pancreas with activated β-catenin. The upregulation of the Hh pathway in embryonicpancreas inducedby β-catenin activation is in concordance with previousstudies describingthe effectsof overactivating the Hh pathway in the pancreas [37]. Thus, misexpression of the Hh ligand Shh in the pancreatic domain causes a pancreatic to intestinal transformation reminiscent of pancreata with activated β-catenin, including increased SMA-positive mesenchyme and epithelial cysts. Also, in vitro studies performed in pancreatic rudiments have shown that activin A causes differentiation of intestinal tissue from embryonicpancreas via a Hh-dependent mechanism [23]. Thus, our study providesa mechanistic frameworkfor our and previously reporteddata regarding the role of these signalingpathways in gut development. Our results provide additional evidencefor the remarkable developmental plasticity of pancreatic progenitors [4]. Other studies have shown cell fate switches of the pancreatic epithelium, including pancreatic to intestinal [37,45–47] and pancreatic to liver transformation [48]. However, this plasticity seems to be limited to the early stages of pancreas development. Activation of β-catenin signalingafter E12.5 does not impair pancreas formation [11]. Although expression of gastricand intestinal markers was not specificallyevaluated in these studies, thelack of morphologicalabnormalities in thosepancreata indicate that the pancreatic epithelium is refractoryto the effectof β-catenin in activation of the gastrointestinal geneticprogram. Interestingly, a similar time window for plasticity of the pancreatic epithelium has beenreportedfor pancreas-to-liver transformation inducedby loss of the transcription factor Sox9 [48]. This periodcoincides with the stage at which the pancreatic epithelium contains multipotent progenitor cells with the potential to give rise to all pancreatic cell typessuggestingthat this undifferentiatedstate is particularlysensitiveto developmental plasticity. Our study illustrates the importanceof precise spatial and temporal control of the activityof embryonic signalingpathways for proper organ formation. Our work could provide insight into various human developmental anomalies of the pancreas such as pancreatic agenesis and pancreatic cysts [49]. Severalmutations in genes encodingtranscription factors have been associated with human developmental disorders of the human pancreas (reviewedin [1,9]). However, little is known about the possible involvement of embryonic signalingpathways in these developmental anomalies. To this regard, cases of atrophy of the pancreas associated with cysts that are reminiscent of the cysts found in Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice have been reported [50,51]. It would be interesting to analyze whetheractivation of Wnt/β-catenin signalingis observedin these malformations of the pancreas. Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 17 / 22 Supporting Information S1 Fig. Pancreatic abnormalitiesin Pdx1-Cre; Ctnnb1 tm1Mmt/+ newbornmice.(A, D) Whole mount pictures of representative P0 pancreata from Pdx1-Cre; Ctnnb1 tm1Mmt/+ and control embryos.Low magnificationpictures of Hematoxylin/eosin-stained paraffin sectionsof control (B) and Pdx1-Cre; Ctnnb1 tm1Mmt/+ P0 mice (E). The boxed areas in B and E are shown at higher magnification in C, F and G. High magnificationpicture of control pancreas (C). High magnificationpicture of the scarce normal pancreatic tissue (F) and pancreatic cysts (G) in Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice. Immunohistochemistryreveals membranous localization of β-catenin in control pancreatic tissue (C') and in normal pancreatic tissue of Pdx1-Cre; Ctnnb1 tm1Mmt/+ mice (F'). Nuclear β-catenin localization is found in epithelial cells of pancreatic cystic structures of Pdx1-Cre; Ctnnb1 tm1Mmt/+ P0 mice (G'). Reducedislet formation Pdx1-Cre; Ctnnb1 tm1Mmt/+ newbornmice (H) compared to control mice (I). Higher magnificationpictures are shown in insets. (J) Geneticlabelingof pancreatic cells by detectionof reporterβgalactosidaseactivity in newbornP0 Pdx1-Cre; R26R; Ctnnb1 tm1Mmt/+ pancreata. β-galactosi- dase was restrictedto the epithelial compartment.Note that the epithelial cells lining the cysts were positive for β-galactosidase.Scale bars, 100 μm. d, duodenum; dp, dorsal pancreas; Li, liver st, stomach; vp, ventral pancreas. (TIF) S2 Fig. Impairmentof pancreaticdifferentiation in Pdx1-Cre; Ctnnb1 tm1Mmt/+ embryos. Hematoxylin/eosin-stainedparaffinsectionsof E15.5 control (A) and Pdx1-Cre; Ctnnb1 tm1Mmt/+ pancreata (B). (D) ReducedPTF1a expression in E13.5 Pdx1-Cre; Ctnnb1 tm1Mmt/+ embryonic pancreata compared to control embryos(C). (F) ReducedNGN3 expression in E13.5 Pdx1-Cre; Ctnnb1 tm1Mmt/+ embryonicpancreata compared to control embryos(E). Higher magnificationpictures are shown in insets. d, duodenum;dp, dorsal pancreas; p, pancreas; s, spleen; st, stomach; vp, ventral pancreas. (TIF) S3 Fig. Histologicalanalysisof embryonicRfx6-Cre; Ctnnb1 tm1Mmt/+ pancreata.Whole mount pictures of representative P0 pancreata from Rfx-Cre; Ctnnb1 tm1Mmt/+ (A) and control mice (B). Pancreatic tissue is outline in red dashed line. d. duodenum; p. pancreas; s. spleen; stomach;. Immunofluorescence for exocrine markers amylase and mucin reveals normal exocrine formation in the pancreatic remnant of Rfx6-Cre; Ctnnb1 tm1Mmt/+ mice (C, D). Decreased endocrinestructuresin Rfx6-Cre; Ctnnb1 tm1Mmt/+ mice (F) compared to control pancreata (E). Higher magnification pictures are shown in insets. Reducedepithelium in early embryonic Rfx6-Cre; Ctnnb1 tm1Mmt/+ pancreata compared to control pancreata, as revealedby carboxypeptidaseA1 (G, H) and Pdx-1 (I, J) immunohistochemistry. (G) Quantification of Cpa1-posi- tive area in Rfx6-Cre; Ctnnb1 tm1Mmt/+ and control E13.5 embryonic pancreas. Scale bars, 100 μm. (TIF) S4 Fig. Loss of pancreatictissue in Rfx6-Cre; Ctnnb1 tm1Mmt/+ embryos.Representative sequential paraffin sectionsstained for Pdx-1 antibody of Rfx6-Cre; Ctnnb1 tm1Mmt/+ (A-F) and control (G-L) E13.5 embryonic pancreas. d, duodenum; dp, dorsal pancreas; li, liver; p, pancreas; s, spleen; st, stomach; vp, ventral pancreas. (TIF) S5 Fig. Gastricand intestinalmarkersin Rfx6-Cre; Ctnnb1 tm1Mmt/+ embryonicpancreata. Low magnificationpictures of Rfx6-Cre; Ctnnb1 tm1Mmt/+ (D, E, F) and control (A, B, C) E18.5 embryonic pancreas stained for Sox2, Cdx2 and β-catenin. Higher magnificationpictures of Pancreatic Activation of Gastrointestinal Genes Induced by Wnt-Beta-Catenin PLOS ONE | DOI:10.1371/journal.pone.0164714 October 13, 2016 18 / 22 these images are shown in Fig 4. d, duodenum;dp, dorsal pancreas; li, liver; p, pancreas; s, spleen;st, stomach; vp, ventral pancreas. (TIF) S1 Table. Primaryantibodies. (DOCX) S2 Table. Primer sequences. (DOCX) Acknowledgments We would like to thank Konstantin Levitsky at the Institute of Biomedicineof SevilleMicroscopy facility for help with confocal microscopy. We thank Doug Melton and Mike German for providing the Pdx1-Cre and Rfx6-Cre mouse line, respectively. Author Contributions Conceptualization:JLM-B SP MH DAC. Data curation:JLM-B AF-M DAC. Formal analysis:JLM-B AF-M DAC. Funding acquisition: MH AR DAC. Investigation: JLM-B AF-M GH-M SP AR DAC. Methodology:JLM-B AF-M GH-M SP AR DAC. Project administration:DAC. Resources:MMT. Supervision:DAC. Validation: JLM-B AF-M GH-M DAC. 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