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INTRODUCTIONThe mature pancreas consists of a highly branched, tubularepithelial tree-like network, which makes up the bulk of thepancreas and is primarily exocrine in function. Ductal branches arecapped at the tips by acinar cell clusters and are connected to atrunk-like central duct, which shuttles digestive enzymes into theduodenum (Pictet and Rutter, 1972). Pancreatic branches ramify,as they originate within the acini as small ducts lined bycentroacinar cells, followed by intercalated, intralobular and finallyinterlobular ducts – all of increasing caliber (Bonal and Herrera,2008). In recent decades, understanding of the ontogeny of thepancreatic tree has grown tremendously, driven in large part bystudies of endocrine differentiation, as a basis for cell replacementtherapies to treat diabetes (Murtaugh and Melton, 2003; Oliver-Krasinski and Stoffers, 2008; Slack, 1995).

The entire pancreatic tree arises from an endodermally derivedprotodifferentiated epithelium (Cleaver and MacDonald, 2009).Multipotent progenitors (MPCs) located at branch tips give rise tothe vast majority of pancreatic cells, including acinar, endocrineand ductal lineages (Gu et al., 2003; Zhou et al., 2007). Aspancreatic epithelium differentiates during late gestation, acinarcells mature at branch tips, while ductal and endocrine progenitorsemerge in branch stalks. Endocrine cells escape from theirepithelial neighbors via delamination and coalesce into islets. Theremaining tubular epithelium gives rise to the ductal branches thatmake up the pancreatic tree. The exocrine (acinar/ductal) andendocrine compartments thus share their origins within thepre-pancreatic epithelium and grow coordinately, albeit viadramatically different mechanisms, together forming a dual-function integrated organ.

Although a number of studies have provided excellentanatomical models of the early pancreatic bud development(Jorgensen et al., 2007; Pictet and Rutter, 1972), no in depth studiesexist of its early cellular organization or later branching patterns.Shortly after evagination, pancreatic buds present as dense,convoluted epithelial structures. Initiation of branch formation hasbeen hypothesized to occur as a result of random buckling of theendodermal epithelium (Gittes, 2009; Slack, 1995). However, theontogeny of developing pancreatic branches remains poorlyunderstood.

A few reports suggested that pancreatic branch, and thus lumenformation, occurs via ‘microlumen fusion’ within the progenitorepithelium (Hogan, 1999; Jensen, 2004). Recently, an elegant studyreported this unusual process and demonstrated that it requiresproper epithelial cell polarity (Kesavan et al., 2009). However, themechanisms by which the pancreatic epithelium transforms into ahighly branched organ remain to be determined. Understandingepithelial morphogenesis and branching in the developing pancreashas been limited by a lack of descriptive characterization, both atthe macroscopic and cellular levels. As a result, no genetic modelshave been identified that affect these processes, in part due to thelack of an established baseline for comparison.

Here, we provide an anatomical model of pancreatic branchingbased on the analysis of hundreds of developmental intermediates.This model will serve as reference for future comparison andanalysis of mutants, allowing identification of molecules thatregulate pancreatic epithelial morphogenesis and branching. Wedemonstrate that the developing pancreas displays predictabletrends in overall shape and in the elaboration of specific branches.In addition, we propose a new model for pancreatic branching inwhich a multi-lumen epithelial network remodels into single-lumenramifying branches. This process involves a number of cellularevents, including transient epithelial stratification, dynamic cellpolarity shifts, asynchronous apical cell constriction and rosetteorganization, as well as microlumen formation and fusion. Thismechanism of branch formation is novel and in stark contrast toclassically described branching models (Andrew and Ewald, 2009).Significantly, we identify a genetic model in which these processesare affected. We demonstrate that EphB signaling is required for

Development 137, 4295-4305 (2010) doi:10.1242/dev.052993© 2010. Published by The Company of Biologists Ltd

1Department of Molecular Biology, University of Texas Southwestern Medical Center,5323 Harry Hines Boulevard, Dallas, TX 75390, USA. 2Department of DevelopmentalBiology, University of Texas Southwestern Medical Center, 5323 Harry HinesBoulevard, Dallas, TX 75390, USA.

*Author for correspondence (ondine.cleaver@utsouthwestern.edu)

Accepted 30 September 2010

SUMMARYThe mammalian pancreas is a highly branched gland, essential for both digestion and glucose homeostasis. Pancreatic branching,however, is poorly understood, both at the ultrastructural and cellular levels. In this article, we characterize the morphogenesis ofpancreatic branches, from gross anatomy to the dynamics of their epithelial organization. We identify trends in pancreatic branchmorphology and introduce a novel mechanism for branch formation, which involves transient epithelial stratification and partialloss of cell polarity, changes in cell shape and cell rearrangements, de novo tubulogenesis and epithelial tubule remodeling. Incontrast to the classical epithelial budding and tube extension observed in other organs, a pancreatic branch takes shape as amulti-lumen tubular plexus coordinately extends and remodels into a ramifying, single-lumen ductal system. Moreover, ourstudies identify a role for EphB signaling in epithelial remodeling during pancreatic branching. Overall, these results illustratedistinct, step-wise cellular mechanisms by which pancreatic epithelium shapes itself to create a functional branching organ.

KEY WORDS: Epithelium, Stratification, Polarity, Branching, Microlumen, Tubulogenesis, Plexus, Remodeling, Eph, Ephrin, Mouse

Epithelial dynamics of pancreatic branching morphogenesisAlethia Villasenor1, Diana C. Chong1, Mark Henkemeyer2 and Ondine Cleaver1,*

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proper pancreas development, as pancreatic epithelium andpredicted branching patterns are disrupted in the absence of EphBreceptor function.

MATERIALS AND METHODSMice and embryo handlingExperiments were performed in accordance with protocols approved by theUT Southwestern Medical Center IACUC. E8.0-E18.5 CD1 embryos werefixed in 4% PFA/PBS overnight at 4°C, rinsed in PBS and stored at –20°C.EphB2lacZ/lacZ/EphB3–/– were maintained on a CD1 background.Littermates or CD1 staged matched embryos were used as controls.

Whole-mount in situ hybridization and RNA probesExperiments were carried out as previously described (Villasenor et al.,2008). Probes used were: Barx1 (~870 bp, BC119047); ephrin B1 (~800bp); Hox11 (~2067 bp, BC018246); Ngn3 (539 bp); and Sox9 (2305 bp,BC023953) from Open Biosystems. Images were taken using a NeoLumardissecting microscope (Zeiss) and a DP-70 camera (Olympus).

Pdx1-lacZ embryo generation and -galactosidase reactionPdx1-lacZ heterozygous males were mated to CD1 females. Pancreatawere fixed in 4% PFA/PBS for 15 minutes, rinsed in PBS and stained for-gal, as previously described (Villasenor et al., 2008). Images were takenwith a NeoLumar stereomicroscope (Zeiss) using a DP-70 camera(Olympus).

Immunofluorescence of paraffin sectionsSections were deparaffinized and treated with R-Buffer A or R-Buffer B ina 2100 Retriever (Electron Microscopy Sciences); blocked with CAS-Block (Invitrogen); and incubated with primary antibody. Concentrationsof primary antibodies were: rabbit -aPKC (1:500 Santa Cruz); goat --catenin (1:50 Santa Cruz); rabbit -collagen IV (provided by R. Brekken);mouse -E-cadherin (1:200 Invitrogen); rabbit -ezrin (provided by T.Carroll); guinea pig -Glucagon (1:600 provided by R. MacDonald);mouse -GM130 (1:500 BD Biosciences); rabbit -laminin (1:200 Sigma);rabbit -Par3 (1:500 Invitrogen); rabbit -Ptf1a (1:1000 provided by R.MacDonald); and mouse or rabbit -ZO1 (1:50 Invitrogen). Secondaryantibodies were from Jackson ImmunoResearch Labs or Invitrogen. Imageswere acquired on a LSM510 META Zeiss confocal microscope.Fluorescence intensity ratios of -catenin were calculated by intensity ofdelineated cell membrane versus cytoplasm (12 different raw micrographs),within arbitrary areas containing ~15 cells each (both cap and body cells),from three individual buds (three wild type and three EphB mutant) withImageJ software. Student’s t-test was used to calculate P value for allgraphs.

Whole-mount immunofluorescence of pancreatic gutsFixed pancreatic guts were permeabilized with 1% Triton X100/PBS for 1hour, blocked with CAS-Block Reagent (Invitrogen), incubated witharmenian hamster anti-mucin 1 (1:200 Thermo Scientific) overnight at 4°C,followed by a Cy3 secondary antibody. Pictures were taken every 1 mmusing a Zeiss Stereo Discovery. V12 with Pentafluar S. Image J softwarewas used to reconstruct images.

3-D reconstructionsPFA-fixed pancreata were sucrose embedded in optimal cuttingtemperature (OCT) medium. Frozen sections (30 mm) were treated asdescribed above for paraffin sections. Z-stacks were taken every 1 mm withan LSM510 META Zeiss confocal microscope. Three-dimensionalreconstructions were made using Imaris software.

RESULTSAnatomy of the embryonic pancreasTo understand the ontogeny of pancreatic branches, we examinedand compared between 20-50 embryonic Pdx1-lacZ pancreata(Offield et al., 1996) at each stage of embryonic development (Fig.1A-I; 10 shown per stage in Fig. S1 in the supplementary material).We identified common features of the embryonic dorsal pancreas,

defined conserved landmarks not previously noted and created arepresentative anatomical model that summarizes distinct andreproducible trends in pancreatic epithelial morphogenesis (Fig. 1J).

Splenic and gastric lobes of the dorsal pancreas(E12.5-birth)As previously described (Bock et al., 1997), the murine dorsalpancreas is composed of a large splenic lobe and a smaller gastriclobe (Fig. 1J). The splenic lobe extends along the stomach from theduodenum (proximal) to the spleen (distal), making up the bulk ofthe dorsal pancreas. The gastric lobe extends along the posteriorstomach (Fig. 1D-H,J).

The anvil-shaped tail (E12.5-birth)This comprises the distal and largest region of the splenic pancreas(Fig. 1C-H,J). Termed ‘tail’, in analogy with the distal region of thehuman pancreas (Cleaver and Melton, 2004), it takes on acharacteristic ‘anvil-like’ shape by E14.5. The tail is grosslypyramidal and consists of compound branches.

The ridge (E11.5-birth)This region forms the apex of the pyramid-shaped tail and runs alongthe proximodistal axis of the pancreas (Fig. 1). It separates anarbitrarily designated ‘left’ from ‘right’ side of the splenic lobe, whendorsal pancreas is viewed laterally (with stomach behind) (Fig. 1J,K).Ridge is initially recognizable around E11.5 (Fig. 1J), and isprominent from E14.5 (Fig. 1M) into postnatal stages (Fig. 1L).

The heel (E13.5-15.5)The heel is a transient feature located on the ‘right’ side of the‘anvil-shaped’ tail. It is a thick, rounded cap of mesenchymeoverlying the branching epithelium of the tail, located near thespleen (Fig. 1J).

The identification of ‘ridge’ and ‘heel’ structures was based bothon morphological features and on localized expression ofmesenchymal genes (Fig. 1N-P). In situ hybridization for themRNAs of Hox11 (Tlx1 – Mouse Genome Informatics), Barx1 andephrin B1 (Efnb1 – Mouse Genome Informatics) at E14.0 revealsmesodermal subdomains within the pancreatic tail. Barx1 marksthe ‘left’ side of the pancreas (Fig. 1N), Hox11 marks the ‘heel’only (Fig. 1O), and ephrin B1 marks the ‘right’ side of the pancreas(Fig. 1P).

Lateral branches (E13.5-birth)The pancreas displays ‘lateral’ type branching (Metzger et al.,2008; Puri and Hebrok, 2007). The splenic lobe displays numerousparallel lateral branches on both sides of the ridge, along itsproximodistal axis. Long lateral branches extend towards theposterior region of the stomach, on the ‘left’ side (Fig. 1E-H,J),while shorter ones are found on the ‘right’, extending towards thestomach fundus (Fig. 1D-H,J).

Ventral pancreas morphology also displayed recognizable trendsof growth during development, including similar patterns and onsetof branching (see Fig. S2 in the supplementary material). However,this study focused on the dorsal pancreas.

Trends in pancreatic branching patternsTo recognize branching patterns and trends, pancreata werecategorized based on their most easily distinguishable features,such as tail morphology and left lateral branches. The relativefrequency of each category was calculated and representativeexamples assembled (see Fig. S3 in the supplementary material).

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We began our analysis at the earliest stage of pancreasdevelopment. As described by others (Jorgensen et al., 2007;Offield et al., 1996; Slack, 1995) E9.5 buds appeared in wholemount as ‘fin-like’ epithelial evaginations on both the dorsal andventral sides of gut tube (see Fig. S3A in the supplementarymaterial) and by E10.5, the dorsal bud appeared ‘fist-like’ (see Fig.S3B in the supplementary material). At these early stages, allpancreata displayed nearly identical gross morphology (see Fig.S1A,B in the supplementary material).

By E11.5, the dorsal bud elongated into a teardrop-shaped structure(see Fig. S3C in the supplementary material) and two forms of thedistal tail became distinguishable: blunt or bifurcated. At this stage,branches were not distinguishable as the bud surface was relativelysmooth. However, by E12.5, the bud surface became completelycovered by stubby, MPC-containing ‘tips’ (Fig. 2A). Throughout thisreport, we will term these structures ‘tips’ for simplicity.

Between E12.5-13.5, bona fide branch formation initiation couldbe discerned. By E13.5, pancreatic ‘branches’ appeared as wideprotrusions, studded with ‘tips’. Of note, at this stage, the patternof nascent branches presented the highest level of variation,compared with all other stages (Fig. 2B� and see Fig. S3 in thesupplementary material). We speculate that this high level ofobserved variation results from the proliferative burst known tooccur during the secondary transition, a timeframe when thepancreas is rapidly changing.

From E13.5 to E17.5, the overall morphology of individualpancreata progressively converged in appearance. Althoughvariability remained at the level of single branches, pancreata werereadily categorized based on their left lateral branches: type I,no/short left lateral branches; type II, one or two branches midwayalong the pancreatic axis; type III, many or long lateral branches(see Fig. S3E-I� in the supplementary material); and a few pancreasat E13.5 and E14.5 displayed unclassifiable pancreatic morphology(~0-10%, data not shown). By E18.5, pancreas morphology fellinto two principal categories: ‘short’ left-branches (Fig. S3J� in thesupplementary material) and ‘long’ left-branches (see Fig. S3J� inthe supplementary material). Morphological features (describedabove) were clearly identifiable in perinatal pancreas (Fig. 1K,L).Thus, whereas pancreatic branching had been thought to berandom, we show that, at least grossly, there is predictablepatterning of branches.

Branch formation: epithelial/ductal plexusremodelingTo understand initiation of branch formation, we examined the firstexternally apparent ‘tips’, around E12.5 (Fig. 2A,A�). Given that‘tips’ consist of MPCs (Zhou et al., 2007), the presumption is thatthey generate extending tubular branches. We thus expected branchformation to result from perpendicular elongation of individualepithelial ‘tips’ (model I, Fig. 2E). Surprisingly, however, we did

4297RESEARCH ARTICLEEpithelial dynamics of the branching pancreas

Fig. 1. Morphological landmarks throughout development of the dorsal pancreas. (A-H)-Galactosidase staining of Pdx1-lacZ dorsalpancreata at several stages of development (E9.5-E18.5, lateral view). (A,B)Anterior is leftwards, dorsal is upwards; (C-H) distal is upwards, proximalis downwards. (I)Graph indicates dorsal pancreatic growth. Data are mean±s.e.m. (J)Anatomical models showing key morphological features.(K)-Gal staining of E18.5 Pdx1-lacZ pancreas. (L)P14 pancreas showing that key features persist into postnatal stages. Ridge is indicated by thedotted white line; right branches are indicated by the dotted grey line. (M)Top view of the anvil-shaped tail of Pdx1-lacZ pancreas. Epithelium isblue; mesenchyme is white. The tail is triangular; the ridge is the apex (red arrow). (N-P)In situ hybridization of E14.0 dorsal pancreata showingheterogeneity of the mesenchyme. (N)Barx1 is concentrated on the ‘left’ side of the pancreas; (O) Hox11 is localized on the ‘heel’, whereas (P)ephrin B1 is enriched on the ‘right’ side. Red arrows in C,J (left) indicates ‘ridge primordium’; red arrows in D-H,J (middle and right),L,M indicate theridge. Brackets in E-H,J,L indicate left lateral branches; dotted lines in C-H,J outline the anvil-shaped tail. (D-H,J) Blue arrow indicates gastric lobe.(E-H,J) Brackets outline left lateral branches. (E,F,J) Triple black arrows, right lateral branches. dp, dorsal pancreatic bud; llb, left lateral branches; rlb,right lateral branches; s, spleen; st, stomach; v, ventral pancreatic bud. Scale bars: 100mm in A-E,M-P; 200mm in F-H; 400mm in K,L.

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not observe ‘tips’ of intermediate and variable lengths, suggestingthere was no progressive ‘tube-like’ extension of ‘tips’. These shortepithelial structures instead remained of fixed and uniform in sizethroughout pancreatogenesis, but increased in number over time(Fig. 2A�-D�; data not shown).

Indeed, at E13.5, left lateral branches appeared to form fromwide lateral protrusions of epithelium that narrowed over time (Fig.2B-D). This morphology suggested that branches formed via

growth and remodeling of the epithelium, rather than by outgrowthof ‘tips’. Our observations thus favor an alternative model forbranch growth in which the contribution of ‘tip’ MPCs to branchgrowth is via longitudinal growth, where ‘tips’ repeatedly divide togenerate new ‘tips’, as well as intervening epithelium, resulting inoverall extension of the underlying branch epithelium (model II,Fig. 2F).

Because the dynamics of pancreatic branch growth wereunexpected, we investigated the emergence of branches from adifferent perspective: examining branch lumens using mucin 1(Muc-1) staining (Pierreux et al., 2006) (Fig. 3). Surprisingly, wefound that prior to appreciable branch appearance, the pancreaticepithelium had already established a complex internal network oflumens, or luminal ‘plexus’ (Fig. 3A,E). This plexus then remodelsto a hierarchical system of tubes (Fig. 3E-G). Interestingly, pre-branch epithelial protrusions contained multi-lumen plexi. As theseprotrusions refined into branches, their multi-lumen networksprogressively remodeled into single lumens (Fig. 3D). Pancreaticbranch lumens therefore developed before any external evidence ofbranching and the multi-lumen tubular plexus coordinatelyextended and remodeled into a ramifying single-lumen tree (Fig.3A-C). To our knowledge, this is the first evidence for this type ofepithelial tube and branch formation.

The pancreatic epithelium: transient stratificationand de-stratificationTo investigate the cellular basis of this unusual branchingmorphogenesis, we assessed the dynamics of pancreatic epithelialorganization during development (Fig. 4). Using E-cadherinimmunofluorescence, we determined that the pancreas arises froma single-layered epithelium that experienced a rapid but transientstratification, and later resolved back to a single-layered state astubes and branches were generated.

Prior to budding, between E7.5-8.25, the pre-pancreaticendoderm is composed of a single layer of cuboidal epithelial cells(Fig. 4A). At these stages, the pre-pancreatic epithelium exhibitsno morphological signs of organogenesis. As the embryo turns,however, we found that dorsal bud epithelial cells transformedfrom cuboidal to columnar, forming a ‘placode’ or epithelialthickening (Fig. 4B). Although this placode was initially a singlecell layer thick, it stratified over the next 2 days, generatingmultiple layers of cells between the duodenal lumen and the outerbasement membrane (Fig. 4B-D,I,J). As development proceeded

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Fig. 2. External branch morphology and growth duringpancreatic development. Morphology of individual branches. Distal isupwards, proximal is downwards. (A-D)-Galactosidase staining ofE12.5-E15.5 Pdx1-lacZ dorsal pancreas. (A�-D�) Higher magnificationdetail of MPC-containing epithelial ‘tips’ covering the bud surface(boxes). ‘Tips’ remain of relatively uniform size (red brackets) duringdevelopment, but increase in number, while branches narrow.(E,F)Models for ‘tip’ contribution to branch growth: (E) model I,epithelial ‘tips’ undergo perpendicular tubular growth, lengthening intoindividual branches; (F) model II, epithelial ‘tips’ undergo longitudinalgrowth by repeatedly dividing, generating new ‘tips’ and generatingoverall extension of the underlying branch epithelium. Blue arrowsrepresent vectorial growth. Scale bars: 100mm in A-D; 50mm in A�-D�.

Fig. 3. Lumen morphology during pancreaticdevelopment. (A-C)Whole-mount Muc1immunofluorescent staining of (A) E12.5, (B) E14.0and (C) E14.75 pancreata, showing ductal system.Distal is at the top and proximal is at the bottom.(D)Higher magnification views and cartoons ofpancreatic branches showing plexus remodeling.(E-G)Higher magnification of the ductal plexus inthe proximal region of the pancreas show initiallysmaller lumens (E), widening lumens (F) andinitiation of tubular remodeling (G). Short brightbranches are small ducts within pre-acinar ‘tips’.Arrows indicate plexus; arrowheads indicateremodeled branches. Scale bars: 50mm.

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through ‘protodifferentiated’ stages (E8.75-11.5), the number ofcell layers increased to approximately six to eight thick (Fig. 4K),surrounding a primary central lumen (PCL) (see Fig. S4 in thesupplementary material).

Of note, we consider all cells within this multilayered bud to be‘epithelial’ in nature, as they express E-cadherin and do not expressmesenchymal markers, such as vimentin (data not shown). High-resolution 3D reconstructions and z-stack movies revealed that,within the most highly stratified region of the E10.5 bud, inner cellscontacted neither the PCL nor the peripheral basement membrane,either directly or via thin extensions (Fig. 4J and data not shown).

As the pancreas progressed through the secondary transition, themulti-layered pancreatic epithelium began to resolve and toprogressively decrease in thickness (de-stratified) as branchesformed (Fig. 4E-H). This resolution was the result of two majorcellular changes: (1) Epithelial cells rearranged to form single celllayered epithelial tubes; and (2) The ductal network remodeledfrom a multi-dimensional plexus to a hierarchical ramifying tree.Interestingly, these changes were concomitant with thedifferentiation of protodifferentiated cells into the differentpancreatic cell lineages: endocrine cells, acini and ducts.

Pancreatic epithelial polarity dynamicsTo further understand how the pancreatic epithelium generatedorganized tubes, we examined the dynamics of cell polaritythroughout stratification and de-stratification (Fig. 5). Although thepancreatic bud originated from a typical polarized epithelium (datanot shown), during stratification some of these epithelial cellspartially or completely lost their polarity. This was a transientphenomenon, as these cells recovered typical polarity when theepithelium resolved back to a single layer and branches formed.

Once fully stratified, at E10.5, pancreatic epithelial cellsorganized in a manner similar to the mammary gland (Lu andWerb, 2008; Mailleux et al., 2008; Sternlicht, 2006), with at leastthree recognizable domains: an outer layer of ‘cap’ cells, interim‘body’ cells and innermost ‘lumen-lining’ cells (Fig. 5H). ‘Cap’cells maintained their basal polarity, as they directly contacted thebasement membrane (laminin and collagen on basal surface, basalnuclear localization) (Fig. 5A,B), but displayed no apical polarity,as they lost expression of the apical PAR complex (APKC, Par3)(Fig. 5C-D) or ezrin (Fig. 5E). ‘Body’ cells also displayed no apicalmembrane domain, as assessed by the absence of aPKC; however,they displayed little basal polarity, as assessed by laminin orcollagen IV deposition (Fig. 5A,B; data not shown). The lumen-lining cells, in contrast to cap cells, showed only apicalpolarization, as they exhibited direct contact with luminal spaceand significant accumulation of all apical markers, including aPKC,Par3, ezrin and ZO-1 (Fig. 5A-E, and data not shown), but no basalpolarization.

Loss of polarity in the stratified pancreatic epithelium wastransient and step-wise. The first sign of reacquired polarity in bothcap and body cells was apical localization of ZO-1, as early asE10.5 (Fig. 5F�; Fig. S5 in the supplementary material). At thistime, however, no other apicobasal markers were detected in bodycells. Of note, we frequently observed single body cells thatharbored ZO-1 on multiple sides (see Fig. S5B,F in thesupplementary material). In addition, cap cells became elongatedover time, exhibiting basal nuclear localization (Fig. 5F) and apicallocalization of the Golgi (GM130) (Fig. 5G).

The progressive re-emergence of complete apical and basalpolarity was temporally associated with the secondary transition.Polarized, monolayered epithelial cells reemerged during this time

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Fig. 4. Pancreatic epithelial stratification and resolution during pancreatic budding from E7.5 to E15.5. (A-H)Immunostaining of E-cadherin on (A-C) transverse sections and (D-H) sagittal sections of dorsal pancreatic buds throughout development. Sections in A-E showpancreatic stratification, while sections in F-H show de-stratification of the pancreatic epithelium. White arrows indicate single cuboidal epitheliumlayer. Pink arrows indicate newly formed acini. Dotted pink line delineates pancreatic ‘placode’ of columnar epithelium. Dotted white line delineatesepithelium of dorsal bud. Dotted green lines outline endocrine clusters. Yellow asterisk marks PCL. (I)Higher magnification image and cartoon ofdetail in B, showing a mostly single-layered epithelium. (J)Higher magnification image and cartoon of detail in D, showing epithelial stratification.(K)Graph showing average number of epithelial layers increases (stratification) and then decreases (resolution) during development. g, gut tube, nf,neural folds. Scale bars: 25mm.

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(Fig. 5A�-E�,H), while gradually fewer stratified body cellsremained (Fig. 5A�-E�). At about E15.5, almost all cells within thebranched epithelium displayed full apicobasal polarity and wereintegrated into a single-layered branched epithelium (data notshown).

Microlumen formation and tubulogenesisTo characterize luminal network formation, we examined Muc1staining of the pancreatic bud (Fig. 3). The PCL, an initiallyprominent central cavity within the bud (Fig. 4D), quicklythinned as the bud elongated and de novo lumens emerged. Thelate E10.75 bud therefore consisted primarily of a formingplexus (Fig. 6A). At this stage, multiple small isolated lumenswere evident, as well as networks of fusing lumens (Fig. 6B).Serial z-stack reconstructions showed that, over time, isolatedlumens integrated into the tubular network. Connection ofmicrolumens to each other appeared to occur via ‘polarizedcanals’, or tracts of polarized epithelial cells that presaged futurelumens (Fig. 6C).

Analysis of adjacent serial sections revealed that body cellsunderwent dramatic cell shape changes and rearrangements duringlumen formation. Most body cells at E10.5 initially lacked obviouspolarity. However, by E10.75 they quickly reacquired polarity, asassessed by re-emergence of ZO-1 immunoreactivity (see Fig. S5

in the supplementary material). Single body cells often displayedmultiple foci, or aggregates, of ZO-1 expression at this stage (Fig.6F,G).

In some single cells, expression of ZO-1 was distinguishable in anO-ring pattern, forming an apical ‘collar’ (Fig. 6D,E). Whenfollowing single cells across serial sections, we determined that O-rings represented the constricted apical side of a bottle-shaped bodycell, within the stratified epithelium (see Fig. S6 in thesupplementary material). These cells appeared to have undergoneasynchronous apical constriction within the multilayered epithelium,as they appeared in no specific order and in random locations.

As development progressed, we noted the increasing propensityof bottle-shaped body cells to form distinct ‘rosettes’ with othersimilarly shaped body cells (Fig. 6F-I). These rosettes of newlypolarized bottle cells oriented their ZO-1 expressing apical ends atthe center. Rosettes appeared to ‘open up’ isolated microlumensbetween them (Fig. 6I). Such isolated epithelial ‘pockets’ werereadily detected in 3D reconstructions and appeared toprogressively connect to each other during development, formingcontinuous lumen networks (Fig. 6C,J).

Taken together, these data demonstrate that branch formation ispreceded by epithelial stratification, cell shape changes and polarityshifts, microlumen formation and fusion, followed by tubularplexus remodeling into the tree-like system of pancreatic branches.

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Fig. 5. Loss of cell polarity during stratification and resolution during branch formation. Immunofluorescent staining on sagittal pancreaticsections of E10.5 (top panels) and E13.5 (middle panels) as indicated. (A-E)Arrows indicate lumens; brackets indicate body cells. (A�-E�) Arrowsindicate epithelial cells displaying clear apicobasal polarity and asterisks indicate unpolarized body cells. (F)DAPI staining of E11.75 pancreatic bud.Arrows in cap cells indicate basal nuclear positioning. (F�)Same section as F, stained for ZO-1 and -catenin. Arrows indicate polarized localizationof ZO-1. a, columnar cap cell; b, rounded body cell. (G)Laminin and GM130 show polarization of cap cells. Inset shows lower magnification view.Broken line, pancreatic epithelium, except in A,A�,B,B’ where it is outlined by laminin or collagen IV. (H)Schematic, of cellular domains withinstratified pancreatic epithelium (E10.0-E12.5) and resolving bud (E12.0-E15.5). Scale bars: 25mm in A-E,A�-E�; 10mm in F,F�,G.

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EphB signaling is required for proper pancreaticbranching and developmentEphB2 and EphB3 receptors are expressed in the pancreaticepithelium (see Fig. S7 in the supplementary material) (van Eyll etal., 2006) and the compound EphB receptor mutant miceEphB2lacZ/lacZ/EphB3–/– display defects in epithelial organization inthe thymus and intestine (Batlle et al., 2002; Garcia-Ceca et al., 2009;Miao and Wang, 2009; Munoz et al., 2009). However, to date,pancreas structure has not been evaluated in these mutants. Using ourcriteria for normal pancreatic development, we assessed whetherEphB signaling was required for pancreatic epithelial developmentand branching.

To visualize pancreas epithelium and gross morphology inEphB2lacZ/lacZ/EphB3–/– mice, we assayed Ngn3 and Sox9expression (Villasenor et al., 2008). Mutant pancreata displayeddefects in overall morphology and branching, including a reductionin size and thickness of both splenic and gastric lobes, and loss ofthe characteristic ‘anvil-shaped’ tail (Fig. 7A,B,G). The lengths ofmany left lateral branches in the distal region of the dorsal pancreaswere severely reduced in mutants and the ‘ridge’ was greatlyflattened (Fig. 7C,D). In addition to branching abrogation, theregionalization of mesenchymal genes was also lost (see Fig. S8 inthe supplementary material), indicating crosstalk betweenmesenchyme and epithelium is probably required for properpancreatic development. The penetrance and severity of the mutantphenotype was variable, with 54% of the total number ofEphB2lacZ/lacZ/EphB3–/– displaying disrupted morphology and 30%exhibiting severe phenotypes (similar to mutants in Fig. 7).

Disrupted branching morphology was present at later stages inmutants and was accompanied by decreased exocrine mass (datanot shown). E16.5, P14 and adult mutant pancreata were smaller,displaying shorter branches and reduced tails (Fig. 7E,F; data notshown). In addition, although adults were viable and fertile, theyhad lower body weights, displayed smaller less branched pancreataand exhibited defects in glucose homeostasis (data not shown).

To discern whether the observed phenotype might be due to asingle EphB receptor, we examined EphB single mutants. TheEphB2lacZ/lacZ/EphB3–/– mice are null for the EphB3 receptor anddominant negative for the EphB2 receptor. The cytoplasmicdomain of EphB2 is replaced with -galactosidase, therebyblocking forward signaling but allowing reverse signaling via Ephligands. To determine which receptor was required for properbranching, the compound mutant EphB2lacZ/lacZ/EphB3–/– wascompared with the single mutants EphB3–/– and EphB2lacZ/lacZ.Neither of the single mutants showed defects in pancreatic grossmorphology or branching (see Fig. S9 in the supplementarymaterial), suggesting that both EphB2 and EphB3 receptors arerequired coordinately for pancreatic development.

EphB2lacZ/lacZ/EphB3–/– mutants display disruptedepithelial organization and lumen formationTo visualize lumens in wild-type versus EphB2lacZ/lacZ/EphB3–/–

mutants, 3D reconstructions of Muc1 immunostained pancreatawere examined at different stages (E12.5, E14.5, P1). At E12.5,mutant lumens were initially narrower (collapsed) compared withwild type (see Fig. S10A-D in the supplementary material). By

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Fig. 6. Lumen formation occurs as epithelial cells regain polarization and undergo apical constriction. (A)Whole-mount Muc1 staining ofE10.75 pancreatic bud. (B)Higher magnification view of A shows isolated microlumens (blue arrows) and connecting polarized canals (pink arrows).(C,D)Immunofluorescent staining of E12.5 pancreatic epithelium of ZO-1 and E-cadherin. (C)Merged z-stack image of 30mm E12.5 pancreatic section.Arrow indicates pre-lumen polarized canal. (D)Higher magnification view of E12.5 section showing ZO-1 ring (yellow arrow) and bottle cells (asterisk).(E)Cartoon of bottle cells with apical constriction and a ring of ZO-1. (F-I)Serial sections of E12.0 dorsal pancreatic bud stained for ZO-1 and -cateninprotein. (F)Unpolarized epithelial cells exhibit dispersed localization of tight junctions. (G)ZO-1 of adjacent cells clusters apically and forms a centralaggregate between cells. (H)Epithelial cells organize around ZO-1 aggregates in a rosette fashion. (I)Microlumen opens and ZO-1 marks apical cellsurfaces. Arrows indicate ZO-1 aggregates. Rosettes are outlined in white. (J)Cartoon schematic of lumen formation at different stages. (K)Cartoonmodel of lumen network formation and remodeling into a tree-like ductal system. D, distal; P, proximal. Scale bars: 50mm in A; 10mm in B; 5mm in C-I.

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E14.5, narrow lumens opened up, but the ductal system failed tocompletely remodel into its normal hierarchical arrangement (Fig.7H,I,J; see Fig. S10E-H in the supplementary material). By P1,reduced expression of Muc1 in the large ducts of EphB mutantsprecluded analysis of the full extent of remodeling (see Fig. S10I,Jin the supplementary material). Together, the data suggests that theluminal system in EphB mutants is delayed in its progression oftubulogenesis and remodeling.

Disorganization of the mutant epithelium was also noted whencompared with wild type (Fig. 7L,M). Mutant pancreaticepithelium exhibited a marked decrease in membrane-associated -catenin, especially in the outer cap cells (Fig. 7K-O; see Fig. S11in the supplementary material). We note, however, that within bodycells, -catenin was often at higher levels in irregular aggregates(see Fig. S11 in the supplementary material). These data suggestthat Eph signaling is required for both proper levels and cellular

localization of -catenin. In addition, as -catenin is a componentof adherens junctions (Gottardi and Gumbiner, 2001), we examinedepithelial junctional complexes in EphB2lacZ/lacZ/EphB3–/– mutants.A similar decrease in junctional E-cadherin was evident (Fig.7P,Q), suggesting a fundamental change in the structural cohesionof mutant epithelium. Accordingly, sections revealed disorganizedrosettes (Fig. 7R,S) and confirmed early lumen ‘collapse’ (Fig.7T,U).

As -catenin influences growth of the pancreas (Dessimoz et al.,2005; Murtaugh et al., 2005; Wells et al., 2007), we examinedwhether MPCs or progenitor epithelium might show earlyreduction in EphB mutants. Although the number of CPA+ cells atE11.5 were relatively unchanged, the number of Ptf1a-expressingtip cells (Masui et al., 2008) were significantly decreased (Fig.7V,W; see Fig. S12 in the supplementary material) As Ptf1a+

epithelium gives rise to all pancreatic lineages (Kawaguchi

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Fig. 7. Branching defects observed when EphB signaling is impaired. (A-D)In situ hybridization of Sox9 in E14.5 dorsal pancreata.(A,B)Abnormal pancreatic morphology in Eph mutant. (C,D)Top views of pancreatic tails showing a decrease in the ridge height in Eph mutant.(E)Wild-type and (F) EphB mutant E16.5 pancreas showing abrogated distal tail morphology in the mutant. Compare widths of tails (red brackets).Broken line outlines pancreatic epithelium. (G)Graphs measuring differences in tail area and tail ridge height in E14.5 wild-type and EphB mutant.Results are mean±s.e.m. (H-J)Whole-mount Muc1 staining and tracings showing a more primitive ductal plexus in the Eph mutants at E14.5.(K)Quantification of -catenin levels. Immunostaining of E12.0 dorsal pancreata of (L,M) ZO-1 and -catenin, showing disorganized epithelium inthe mutant. Arrow indicates lower levels of -catenin. (N,O)Higher magnification of -catenin protein in wild-type and EphB mutant epithelium.(P,Q)Higher magnification of E-cadherin immunostaining, showing lower levels at the membrane in EphB mutants at E12.0. (R,S)High-magnification view of epithelium (ZO-1 and -catenin) showing reduced rosettes (outlined in white) and apical increase in ZO-1 expression in EphBmutants. Arrowheads indicate microlumens. (T,U)High magnification of a microlumen in wild type versus EphB mutant at E12.0. ZO-1, green; -catenin, red. (V,W)Immunostaining of pancreatic bud showing a reduced Ptf1a-expressing epithelium in EphB mutants. WT, wild type; Eph mut,EphB2lacZ/lacZ/EphB3–/–. Scale bars: 50mm in A-D; 100mm in E,F; 10mm in H,I,R,S; 25mm in L,M,V,W; 5mm in N-Q,T,U.

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et al., 2002), we examined endocrine numbers. Indeed,EphB2lacZ/lacZ/EphB3–/– pancreata developed significantly fewerendocrine cells throughout development and into adulthood (datanot shown). Interestingly, this decrease was more prominent in thedistal region of the tail where most branching defects were found.Together, these results demonstrate a previously unreported role forEphB signaling in pancreatic epithelial development and branching,via proper -catenin levels and cellular localization.

DISCUSSIONIn this study, we provide an in depth analysis of pancreaticbranching that includes both an ‘external’ analysis of the overallbranch morphology and an ‘internal’ analysis of the pancreaticepithelium and its developing tubular network. It has long beenthought that the pancreas displays random branching patterns(Slack, 1995); however, our observations demonstrate predictablebranching trends. Furthermore, our analysis elucidates novelcellular dynamics that underlie pancreatic branching. We showdramatic transient stratification of bud epithelium, in whichmicrolumens form as a result of cell shape changes and fuse to oneanother to form a luminal plexus. This complex network of tubesthen undergoes remodeling to later generate pancreatic branches.We also demonstrate genetic regulation of the branching program,as compound EphB receptor signaling mutant mice display defectsin epithelial organization, a decrease in pancreatic progenitorepithelium, a delay in normal tubular remodeling and aconcomitant reduction in pancreatic branching. Together, this studyestablishes a baseline reference for future identification ofpancreatic regulatory genes and it reveals novel aspects ofepithelial organogenesis.

Stratification and polarity in the pancreaticepitheliumFormation of pancreatic branches and their lumens is preceded, inboth the dorsal and ventral buds, by epithelial stratification andshifts in cell polarity. Interestingly, the process of stratificationcoincides with the ‘protodifferentiated state’ in pancreas, when thepool of multipotent progenitors is expanding (Cleaver andMacDonald, 2009). The epithelium then resolves, as the progenitorpool diminishes and cells differentiate into endocrine, exocrine andductal lineages. It is possible that transient stratification is integralto accumulating the proper pool of progenitors required to give riseto the mature pancreas.

Once a pool of unpolarized body cells has accumulated, thesecells almost immediately begin to reacquire polarity and rearrangeto form microlumens. The first evidence of polarity re-establishment within stratified epithelium is the emergence of theapical tight junction component ZO-1 and of the apical/lumenmarker Muc1. Proper localized accumulation of these twomolecules and other apical proteins in pancreatic stratifiedepithelium has been shown to depend on Cdc42 function (Kesavanet al., 2009). It is only later during lumen formation that standardpolarity molecules such as aPKC accumulate, followed by ezrin,Par3/6 and laminin in resolving branches.

During reacquisition of polarity by body cells (E10.0-10.75),individual cells experienced asynchronous apical constriction, eachforming a tight collar of ZO-1. Localized ZO-1 in single cells isfollowed by cell shape changes in neighboring cells, resulting inthe formation of a rosette of cells with apical ends gatheredtogether between them. Interestingly, focal ZO-1 aggregation inbody cells and rosette formation closely resembled development ofthe intestinal lumen in zebrafish (Horne-Badovinac et al., 2001). In

both cases, initially scattered junctions between epithelial cellsundergo reorganization, forming small foci at cell-cell interfaces.These foci merge with one another until there is a single cluster atthe center of each rosette, allowing lumens to emerge at the centerof these highly polarized clusters. We speculate that pancreaticlumens emerge as proposed in the zebrafish gut lumen, includingrearrangement of cell junctions and expansion of apical membranedomains.

Although the cellular mechanisms underlying pancreaticstratification are unknown, it is conceivable that initially cap cellsundergo asymmetric cell division, in a manner similar to skinstratification (Lechler and Fuchs, 2005). It will be interesting toelucidate the cellular process of pancreatic epithelial stratification,to identify origins and mechanisms underlying body cell generationand polarity shifts, and to determine whether the plane of divisionhas an impact on cell fate.

Interestingly, the stratified pancreatic bud displays similarities tothat of early mammary and salivary glands (Mailleux et al., 2008;Sternlicht, 2006; Tucker, 2007; Walker et al., 2008). All threegenerate transiently stratified epithelium and form lumens de novo.However, the underlying mechanisms of tubulogenesis and branchformation are likely to be different. Both mammary and salivarygland lumens are formed by apoptotic clearance of body cells(Mailleux et al., 2008; Tucker, 2007). By contrast, pancreaticlumens form independently of apoptosis (data not shown). Inaddition, in the mammary and salivary gland, de novo lumens formafter the organ has already branched, whereas in the pancreas thereverse is true: a ductal plexus appears before branching (Hogg etal., 1983; Mailleux et al., 2008; Sternlicht, 2006; Tucker, 2007).

Pancreatic branching results from ductal plexusremodelingOur work thus demonstrates a new mechanism of branch formationthat occurs in a highly unusual manner, from the ‘inside out’, withlumens forming within a thick multilayered epitheliumsignificantly earlier than branches appear. Externally recognizablepancreatic branches emerge later, following complex remodelingof the lumen plexus rather than simple extension of epithelial buds.Although the geometric process underlying this conversion remainsunclear, it is possible that cellular mechanisms such as epithelialconvergent extension or regression are at play.

Pancreatic ductal remodeling is reminiscent of the cellularchanges observed during blood vessel formation (Risau andFlamme, 1995). Embryonic vessels initially form a homogeneousplexus of tubes that subsequently ‘remodels’ into hierarchicalarrangements of large and small arteries and veins. In large part,these events are driven by the hemodynamic flow of blood withinprimitive vessels (Jones et al., 2006). It remains an unresolved issuewhether flow of exocrine secretions in embryonic pre-ductaltubules is significant enough to play any role in pancreatic ductalplexus remodeling.

Although our observations support lateral branching as theprinciple mechanism of pancreatic branching, as observed usinglive imaging of explanted pancreatic buds (Puri and Hebrok, 2007),we refine the concept here, showing it occurs not only by epithelialextension, but in coordination with epithelial remodeling. We alsonote that branch ‘tips’, presumed by the nature of their name torepresent structures that grow by tubular extension, in fact do notgenerate individual branches. Our observations support a differentmechanism by which MPC-containing ‘tip’ structures insteaddivide and multiply, fueling growth of the remodeling epitheliumby a longitudinal contribution.

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Predictable trends in branch patterningOur data also show that developing pancreatic branches displaypredictable trends in morphology. Comparing hundreds ofindividual pancreata, a baseline model of gross branch patterningwas constructed. Although our analysis focused on the dorsalpancreatic bud, the ventral pancreas undergoes a similar branchingprocess, with onset occurring (as the dorsal pancreas) just beforesecondary transition at about E12.5. We note that owing to theunusual sequential nature of internal lumen formation precedingexternal branch formation, patterning of branches could only beassessed starting at E13.5. Analysis of early ‘branching’ patterns(E10.5-11.5) was inherently precluded during ductal plexusformation, as the outer cap epithelium is initially smooth.

An interesting question that arises is how pancreatic branchesacquire their patterning information. We speculate that themesenchyme is a likely candidate signaling tissue, as we observeddistinct regionalization of the mesenchyme and localizedexpression of various factors. Indeed, mesenchymal domains werealtered in EphB mutants (see Fig. S8 in the supplementarymaterial), suggesting reciprocal signaling between mesenchymeand epithelium. Further analysis of branching patterns uponexperimental disruption of these mesenchymal factors will berequired to further our understanding of pancreatic branchpatterning.

EphB signaling is required for epithelialmorphogenesis in the pancreasAs EphB2/B3 receptors are expressed in pre-ductal epithelium (seeFig. S8 in the supplementary material) (van Eyll et al., 2006) andEph/ephrin signaling influences epithelial cell behavior (Holmberget al., 2006), we analyzed pancreatic branching in the absence ofEphB signaling. Indeed, key morphological features of thedeveloping pancreas were defective in the absence of EphBsignaling. We found early disruption of epithelial rosettes andmicrolumens, delay in ductal remodeling, as well as reduced Ptf1a(progenitor epithelium), resulting in later abrogation of pancreaticbranches and reduction of endocrine/exocrine mass. Together, thesedata indicate that branching defects were probably the result ofdefective epithelial morphogenesis, and suggest that differentiationof pancreatic cell lineages depends on proper architecture,remodeling and extension of the initial epithelium into a branchedorgan.

The molecular basis for EphB mutant epithelial defects appearsdistinct from cell polarity regulation. In EphB mutants, althoughZO-1 is increased and disorganized in body cells, it is nonethelesslocalized apically as expected. In addition, morphological defectsobserved are distinct from those in the recently describedpancreatic deletion of Cdc42 (Kesavan et al., 2009). Although thelack of Cdc42 function in the epithelium results in failure ofepithelial integrity and microlumen coalescence, absence of EphBsignaling results instead in epithelial disorganization, affectingsubsequent lumen and branching development.

These defects are probably due to altered epithelial adhesion inEphB mutants. Decrease in junctional -catenin between epithelialcells in EphB mutants may be accompanied by an increasein cytoplasmic -catenin, and possibly -catenin signaling.Mice expressing a constitutively active form of -catenin inpancreatic epithelium, PdxCreearly-catactive, display epithelialdysmorphogenesis, decreased E-cadherin expression andconcomitant defects in exocrine branches (Heiser et al., 2006).Similarly, EphB2lacZ/lacZ/EphB3–/– mutants exhibited decreasedjunctional E-cadherin levels, along with pancreatic hypoplasia and

decreased branching. However, EphB2lacZ/lacZ/EphB3–/– mutantpancreata did not exhibit the cyst formation seen in PdxCreearly-catactive mice (data not shown), reflecting a less severe pancreaticphenotype. Taken together, these data demonstrate that EphBsignaling regulates pancreatic epithelial morphogenesis, includingmicrolumen formation and acinar development, potentially viaregulation of epithelial cell adhesion and -catenin proteinavailability.

A cellular model for pancreatic lumen ontogenyand branchingOur study reports the unusual epithelial dynamics that underliepancreas morphogenesis. It also provides the first analysis ofmurine pancreatic branching and overall morphology duringembryonic development, and therefore sets a baseline for the futureevaluation of pancreatic structure in mouse mutants, for theidentification of regulatory genes. Given that both islet endocrinecells and the entire exocrine glandular tree originate in theprotodifferentiated pancreatic epithelium, elucidating its ontogenyand changing architecture will advance our understanding of thedevelopmental steps important to its cell lineages, both exocrineand endocrine, and their progenitors.

AcknowledgementsWe are grateful to Chris Wright for the Pdx1-lacZ mice; to Thomas Carroll,Raymond MacDonald and Rolf Brekken for reagents; to Jose Cabrera forartwork; and to Courtney Karner and Kate Luby-Phelps/Abhijit Bugde (UTSWLive Cell Imaging core) for their assistance with imaging. We also thank RayMacDonald, JMST and Thomas Carroll for invaluable discussions; and RayMacDonald, Galvin Swift, Eric Olson, Paul Krieg, Michelle Tallquist, StryderMeadows and Jenny Hsieh for critical reading of the manuscript. This workwas supported by grants JDRF Award 99-2007-472 and NIH R01 grantDK079862-01 to O.C. Deposited in PMC for release after 12 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.052993/-/DC1

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