Epithelial dynamics of pancreatic branching morphogenesis

Abstract

The 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 of pancreatic branches, from gross anatomy to the dynamics of their epithelial organization. We identify trends in pancreatic branch morphology and introduce a novel mechanism for branch formation, which involves transient epithelial stratification and partial loss of cell polarity, changes in cell shape and cell rearrangements, de novo tubulogenesis and epithelial tubule remodeling. In contrast to the classical epithelial budding and tube extension observed in other organs, a pancreatic branch takes shape as a multi-lumen tubular plexus coordinately extends and remodels into a ramifying, single-lumen ductal system. Moreover, our studies identify a role for EphB signaling in epithelial remodeling during pancreatic branching. Overall, these results illustrate distinct, step-wise cellular mechanisms by which pancreatic epithelium shapes itself to create a functional branching organ.

Fig. 1.

Morphological landmarks throughout development of the dorsal pancreas. (A-H) β-Galactosidase staining of Pdx1-lacZ dorsal pancreata at several stages of development (E9.5-E18.5, lateral view). (A,B) Anterior is leftwards, dorsal is upwards; (C-H) distal is upwards, proximal is downwards. (I) Graph indicates dorsal pancreatic growth. Data are mean±s.e.m. (J) Anatomical models showing key morphological features. (K) β-Gal staining of E18.5 Pdx1-lacZ pancreas. (L) P14 pancreas showing that key features persist into postnatal stages. Ridge is indicated by the dotted white line; right branches are indicated by the dotted grey line. (M) Top view of the anvil-shaped tail of Pdx1-lacZ pancreas. Epithelium is blue; mesenchyme is white. The tail is triangular; the ridge is the apex (red arrow). (N-P) In situ hybridization of E14.0 dorsal pancreata showing heterogeneity 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 the ridge. 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: 100 μm in A-E,M-P; 200 μm in F-H; 400 μm in K,L.

Fig. 2.

External branch morphology and growth during pancreatic development. Morphology of individual branches. Distal is upwards, proximal is downwards. (A-D) β-Galactosidase staining of E12.5-E15.5 Pdx1-lacZ dorsal pancreas. (A′-D′) Higher magnification detail of MPC-containing epithelial `tips' covering the bud surface (boxes). `Tips' remain of relatively uniform size (red brackets) during development, 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 into individual branches; (F) model II, epithelial `tips' undergo longitudinal growth by repeatedly dividing, generating new `tips' and generating overall extension of the underlying branch epithelium. Blue arrows represent vectorial growth. Scale bars: 100 μm in A-D; 50 μm in A′-D′.

Fig. 3.

Lumen morphology during pancreatic development. (A-C) Whole-mount Muc1 immunofluorescent staining of (A) E12.5, (B) E14.0 and (C) E14.75 pancreata, showing ductal system. Distal is at the top and proximal is at the bottom. (D) Higher magnification views and cartoons of pancreatic branches showing plexus remodeling. (E-G) Higher magnification of the ductal plexus in the proximal region of the pancreas show initially smaller lumens (E), widening lumens (F) and initiation of tubular remodeling (G). Short bright branches are small ducts within pre-acinar `tips'. Arrows indicate plexus; arrowheads indicate remodeled branches. Scale bars: 50 μm.

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 show pancreatic stratification, while sections in F-H show de-stratification of the pancreatic epithelium. White arrows indicate single cuboidal epithelium layer. Pink arrows indicate newly formed acini. Dotted pink line delineates pancreatic `placode' of columnar epithelium. Dotted white line delineates epithelium of dorsal bud. Dotted green lines outline endocrine clusters. Yellow asterisk marks PCL. (I) Higher magnification image and cartoon of detail 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: 25 μm.

Fig. 5.

Loss of cell polarity during stratification and resolution during branch formation. Immunofluorescent staining on sagittal pancreatic sections of E10.5 (top panels) and E13.5 (middle panels) as indicated. (A-E) Arrows indicate lumens; brackets indicate body cells. (A′-E′) Arrows indicate 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 β-catenin. Arrows indicate polarized localization of 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 within stratified pancreatic epithelium (E10.0-E12.5) and resolving bud (E12.0-E15.5). Scale bars: 25 μm in A-E,A′-E′; 10 μm in F,F′,G.

Fig. 6.

Lumen formation occurs as epithelial cells regain polarization and undergo apical constriction. (A) Whole-mount Muc1 staining of E10.75 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 30 μm 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 β-catenin protein. (F) Unpolarized epithelial cells exhibit dispersed localization of tight junctions. (G) ZO-1 of adjacent cells clusters apically and forms a central aggregate between cells. (H) Epithelial cells organize around ZO-1 aggregates in a rosette fashion. (I) Microlumen opens and ZO-1 marks apical cell surfaces. Arrows indicate ZO-1 aggregates. Rosettes are outlined in white. (J) Cartoon schematic of lumen formation at different stages. (K) Cartoon model of lumen network formation and remodeling into a tree-like ductal system. D, distal; P, proximal. Scale bars: 50 μm in A; 10 μm in B; 5 μm in C-I.

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 β-catenin levels. Immunostaining of E12.0 dorsal pancreata of (L,M) ZO-1 and β-catenin, showing disorganized epithelium in the mutant. Arrow indicates lower levels of β-catenin. (N,O) Higher magnification of β-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 β-catenin) showing reduced rosettes (outlined in white) and apical increase in ZO-1 expression in EphB mutants. Arrowheads indicate microlumens. (T,U) High magnification of a microlumen in wild type versus EphB mutant at E12.0. ZO-1, green; β-catenin, red. (V,W) Immunostaining of pancreatic bud showing a reduced Ptf1a-expressing epithelium in EphB mutants. WT, wild type; Eph mut, EphB2^lacZ/lacZ/EphB3^–/–. Scale bars: 50 μm in A-D; 100 μm in E,F; 10 μm in H,I,R,S; 25 μm in L,M,V,W; 5 μm in N-Q,T,U.