The ductal origin of structural and functional heterogeneity between pancreatic islets

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PROGRESS IN HISTOCHEMISTRY AND CYTOCHEMISTRY

Progress in Histochemistry and Cytochemistry 48 (2013) 103–140 www.elsevier.de/proghi

The ductal origin of structural and functional heterogeneity between pancreatic islets Claudia Merkwitz a,1 , Orest W. Blaschuk b,1 , Angela Schulz c , Paul Lochhead d , Jaroslawna Meister c , Angela Ehrlich a , Albert M. Ricken a,∗,1 a

Institute of Anatomy, Faculty of Medicine, University of Leipzig, Liebigstraße 13, 04103 Leipzig, Germany b Division of Urology, Department of Surgery, McGill University, Urology Research Laboratories, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, QC H3A 1A1, Canada c IFB Adiposity Diseases and Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany d Division of Applied Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom

Abbreviations: CK, cytokeratin; E-cadherin, epithelial-cadherin; ed, embryonic day; EP-CAM, epithelial cell adhesion molecule; ew, embryonic week; GCG, glucagon; GHRL, ghrelin; GLP-1, glucagon-like peptide-1; HE, haematoxylin and eosin; HNF1␤, hepatocyte nuclear factor 1 beta; INS, insulin; N-cadherin, neural-cadherin; N-CAM, neural cell adhesion molecule; NGN-3, neurogenin-3; NIC, neuro-insular complex; PC, prohormone convertase; pcd, post coitum day; pp, postpartum; PHHI, persistent hyperinsulinaemic hypoglycaemia of infancy; PPY, pancreatic polypeptide; PDX-1, pancreatic and duodenal homeobox-1; R-cadherin, retinal-cadherin; SOX9, SRY (sex-determining region Y)-box 9; SST, somatostatin; T1D, type 1 diabetes; TGF-␤, transforming growth factor-ß. ∗ Corresponding author. Institute of Anatomy, Faculty of Medicine, University of Leipzig, Liebigstrase 13, 04103 Leipzig, Germany. Tel.: +49 341 97 22 048; fax: +49 341 97 22 009. E-mail address: [email protected] (A.M. Ricken). 1 These authors contributed equally to this work 0079-6336/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.proghi.2013.09.001

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Abstract Islets form in the pancreas after the first endocrine cells have arisen as either single cells or small cell clusters in the epithelial cords. These cords constitute the developing pancreas in one of its earliest recognizable stages. Islet formation begins at the time the cords transform into a branching ductal system, continues while the ductal system expands, and finally stops before the exocrine tissue of ducts and acini reaches its final expansion. Thus, islets continuously arise from founder cells located in the branching and ramifying ducts. Islets arising from proximal duct cells locate between the exocrine lobules, develop strong autonomic and sensory innervations, and pass their blood to efferent veins (insulo-venous efferent system). Islets arising from cells of more distal ducts locate within the exocrine lobules, respond to nerve impulses ending at neighbouring blood vessels, and pass their blood to the surrounding acini (insulo-acinar portal system). Consequently, the section of the ductal system from which an islet arises determines to a large extent its future neighbouring tissue, architecture, properties, and functions. We note that islets interlobular in position are frequently found in rodents (rats and mice), whereas intralobularly-located, peripheral duct islets prevail in humans and cattle. Also, we expound on bovine foetal Laguesse islets as a prominent foetal type of type 1 interlobular neuro-insular complexes, similar to neuro-insular associations frequently found in rodents. Finally, we consider the probable physiological and pathophysiological implications of the different islet positions within and between species. © 2013 Elsevier GmbH. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Primordia (anlagen)-related differences in islet cell composition and function . . . . . . . . Endocrine cells populate the pancreas before islets are present . . . . . . . . . . . . . . . . . . . . . . The cellular origin of the pancreatic endocrine component localizes to the primitive ducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pancreatic endocrine islets continuously arise from cells of the expanding ducts . . . . . . Position-related differences in islet morphology and function . . . . . . . . . . . . . . . . . . . . . . . Laguesse islets are a feature of foetal and neonatal pancreata in cattle, and probably in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comments on studies involving the experimental manipulation of pancreatic islet formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction “The pancreas is a complex tissue and is still perhaps the least understood organ besides the brain in mammals.” (Hennig et al., 2004) There are many excellent reviews concerning the common endodermal origin of pancreatic endocrine and exocrine cells (e.g. Johansson et al., 2007; Bonal and Herrera, 2008; Gittes, 2009). Our review focuses on another less covered and poorly understood topic of

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the endocrine organ component, islet morphogenesis. This process involves topographic reorganization of the endocrine cells to a separate endocrine compartment, the islets, by the end of foetal/neonatal life. We provide a comprehensive overview of our own work, and that of others regarding islet development in humans, rodents (rats and mice), and cattle. Bovine islets are given particular attention for two reasons: firstly, studies have shown that there are more similarities between the embryonic and foetal development of the human pancreas and bovine compact-type pancreata compared to the development of mesenteric-type pancreata in rodents (Doerr and Becker, 1958; Priester, 1974; Goldman et al., 1982; Fowden and Hill, 2001; Piper et al., 2004; Green et al., 2010; Steiner et al., 2010; Merkwitz et al., 2011; Merkwitz et al., 2013); secondly, the islets of cattle and other ruminant pancreata are perfect examples of islet morphological diversification caused by these structures arising from different parts of the ductal system. In particular, we discuss the morphological diversity of islets within and between species. Furthermore, we consider whether insights gained from studying islet function in animal models such as rodents can be applied to humans (even though the islets of the two species differ considerably in origin, architecture, and relationship to surrounding tissue).

2. Primordia (anlagen)-related differences in islet cell composition and function The islet organ is the endocrine component of the vital amphicrine pancreas. It develops together with morphologically distinct exocrine tissue from epithelial cells. These cells are derived from two separate evaginations of the primitive forgut endoderm in close vicinity of the hepatic diverticulum (Slack, 1995; St-Onge et al., 2006). The epithelial cells in the evaginations proliferate, migrate from the gut into the primitive mesodermal mesenchyma of the dorsal and ventral mesogastrium, and branch into cord-like and, subsequently, canaliculated structures, under the influence of signals from the primitive mesodermal mesenchyma and surrounding organs (Villasenor et al., 2010). Lineage tracing has shown that the endodermal founder cells of both endodermal buds (outpouchings) give rise to all mature pancreatic cell types. This is achieved by cell type-specific changes in gene transcription, as well as pre- and post-translational protein modification, and is orchestrated by a cascade of signals and transcription factors expressed in a characteristic sequence (for recent reviews, see Dohrmann et al., 2000; Johansson et al., 2007; Zhou et al., 2007; Desgraz and Herrera, 2009; Rukstalis and Habener, 2009; Mastracci and Sussel, 2012). The two pancreatic primordia (anlagen) locate at the junction of the foregut and the midgut (Tadokoro et al., 2011). This region is sometimes referred to as the hepatopancreatic ring to stress the extraordinary potential of the region to form glandular tissue (i.e. liver and pancreas), and to emphasise that liverand pancreas-like tissues combine into a single organ, the midgut gland, in arthropods and molluscs (van Weel, 1974). The larger dorsal primordium arises first (Table 1), followed by the smaller ventral primordium, which arises initially in paired condition from the posterior end of the hepatic out-pouching, known as the extra-hepatic biliary tree (Spooner et al., 1970; Tadokoro et al., 2011). The cells of the dorsal primordium invade the primitive mesodermal mesenchyma of the dorsal mesentery and are under the early influence of the still doubled aorta and

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Table 1. Developmental time of formation and fusion of rodent, human, and bovine endodermal pancreatic outgrowths. Formation Species

dorsal

mice rats human cattle

*

* ** #

ed 9.5 ed* 11.5 ed* 26 -

Fusion ventral ed* 10.5 ed* 12.5 ed* 28-32 -

ed* 12.5-14.5 ed* 16.5-18.5 ew** 6-7 before pcd# 45

embryonic day embryonic week post coitum day

chorda dorsalis (Lammert et al., 2001). The cells of the right lobe of the ventral primordium invade the primitive mesodermal mesenchyma of the ventral mesentery and come under the influence of the developing heart and diaphragma, while the cells of left lobe subsequently disappear. A developmental rotation in the wall of the primitive forgut soon brings the bile duct and the right ventral pancreatic outgrowth in dorsal contact with the dorsal pancreatic outgrowth, and the two pancreatic outgrowths fuse to form the definitive pancreas (Table 1; Edlund, 1999). The initial anatomical development of the ventral and dorsal primordia are similar, but the signals and transcription factor cascades that control the initial differentiation from pluripotent endodermal precursor cells are different (Gittes, 2009). It is astonishing that the tissue of both endodermal outgrowths finally participates in the establishment of all principal anatomical structures of the mature amphicrine pancreas. The tissue of the ventral outgrowth contributes to most of the proximal duodenal portion of the definitive pancreas (i.e. head including the uncinate process/right lobe), whereas the tissue of the dorsal outgrowth provides the central and splenic portion of the future organ (i.e. body and tail/left lobe). The differing embryonic origins of these pancreatic parts are reflected in the architecture of the lobules, in the temporal appearance of the endocrine components (Fig. 1) and in the cellular composition of the arising islets. In particular, the exocrine lobules differ in size. The lobules also display significant differences in islet number, cellular composition and function (Wittingen and Frey, 1974; Orci et al., 1976; Baetens et al., 1979; Goldman et al., 1982; Fiocca et al., 1983; Sessa et al., 1983; Stefan et al., 1987). It is noteworthy that the ductal system, which is the structure giving rise to the exocrine lobules and endocrine islets, originates from both embryonic outgrowths (see below).

3. Endocrine cells populate the pancreas before islets are present Endocrine cells differentiate in the pancreas from pancreatic and duodenal homeobox-1 (PDX-1) positive progenitors that transiently express the transcription factor neurogenin-3 (NGN-3; Gradwohl et al., 2000; Johansson et al., 2007; Zhou et al., 2007; Desgraz and

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Fig. 1. Confocal photomicrographs of double immunofluorescent-stained paraffin sections (7 ␮m thick) of developing pancreata of 3 cm (A,B) and 5.5 cm (C) crown rump length (CRL) bovine foetuses. A: The part derived from the dorsal primordium (open star) in the early stage pancreas contains a large number of insulin- (green) and glucagon/GLP-1-immunostained (red) cells, whereas the adjacent dorsal primordium part (filled star) contains only a few immunostained cells. B: Higher magnification of A demonstrating the location of hormone-producing cells within the epithelial cords. C: Progression of endocrine cell development from the centre to the periphery of a pancreas from a 5.5 cm CRL foetus. A-C: Note the predominance of glucagon-/GLP-1-immunostained cells (red) among the endocrine cells in pancreata of these early stage foetuses. Scale bars as indicated.

Herrera, 2009; Rukstalis and Habener, 2009). These cells are among the first pancreatic cells encountered in the epithelial outgrowths from the two primordia (Fujii, 1979; Carlsson et al., 2010; Merkwitz et al., 2011). Insulin and glucagon mRNAs can be detected in the murine primitive gut even before the first morphological signs of the two primordia. Thus,

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endocrine gene expression begins in a “pre-morphogenetic phase”, at least in some rodents (Gittes and Rutter, 1992). The pancreatic endocrine cells initially locate as single cells or small clusters of cells between the exocrine precursor cells, along the sides and tips of the epithelial outgrowth (Fig. 1). They then give rise to a morphologically and functionally separate compartment, the endocrine islets (Carlsson et al., 2010; Merkwitz et al., 2011; Merkwitz et al., 2013). Preproglucagon-immunoreactive ␣−cells are the first endocrine cells encountered in the epithelial cords and nests that form the rodent (mouse between ed 9.5 and 10.5, rat at ed 12.5; Rall et al., 1973; Park and Bendayan, 1993; Heller et al., 2004) and bovine primitive pancreata (at pcd 26; Prasadan et al., 2002; Carlsson et al., 2010; Merkwitz et al., 2011; Merkwitz et al., 2013). Significant numbers of other islet hormone-positive cells (such as insulin-positive cells) are typically detected later in these species. Preproglucagon is differentially cleaved by the prohormone convertases PC-2 and PC-1/3 into glucagon and glucagon-like peptides, respectively. Glucagon-like peptide-1 (GLP-1) is a secretion product of the highly specialized endocrine L-cells in the gut, and is known to cause an increase in ␤-cell mass and insulin release in the pancreas (for review see Drucker, 2003). It is likely that GLP-1 may be produced in the early pancreas besides glucagon, and that processing of the prohormone preproglucagon may never entirely switch to glucagon in pancreatic ␣-cells, in contrast to what has previously been believed (Heller and Aponte, 1995; Abraham et al., 2002; Miller et al., 2009; Kilimnik et al., 2010; Marchetti et al., 2012). Proinsulin-immunoreactive ␤-cells subsequently differentiate in the developing rodent and bovine pancreata. This event is followed by the appearance of somatostatin and pancreatic polypeptide (PPY)-producing cells, respectively. The proinsulin-immunoreactive ␤-cells first constitute a small minority of endocrine cells present in the epithelial cords and nests of these species (Fig. 1), but then sharply increase in number to finally constitute the majority of the endocrine cell population in the adult pancreas (Carlsson et al., 2010; Merkwitz et al., 2011). It is particularly noteworthy that the sharp increase in the ␤-cell mass, which is referred to as the secondary transition in rodents (mouse: ed 13.5 to 15.5, rat ed 13.5 to 20.5), coincides with the transformation of the epithelial cords and nests into an expanding branching duct structure leading to the appearance of the first primitive ducts and acini. Surprisingly, several studies have shown that insulin-expressing or somatostatin- and PPY-expressing cells appear first in the developing human pancreas, followed by glucagonand other peptide hormone-expressing cells (Bocian-Sobkowska et al., 1997; Polak et al., 2000; Piper et al., 2004; Jeon et al., 2009; Meier et al., 2010). In addition, some of these studies indicate an unexpectedly high frequency of early endocrine cells concurrently expressing two or three of the islet hormones (Riedel et al., 2012). Other studies report a similar sequence of endocrine cell appearance in humans, as outlined above for rodents and cattle (Peters et al., 2000). This long-standing unresolved controversy concerning the appearance of endocrine cells in humans may result from the use of different testing methods, protocols, and antibody sets, and requires further scrutiny. The abovementioned observations suggest that endocrine cells (at least preproglucagonand proinsulin-immunoreactive cells) are present in the developing pancreas before the exocrine cell differentiation program is initiated, and before endocrine progenitor cells are

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activated to from typical islets. Exclusion of the endocrine progenitor cells from the ductal epithelial lining, and their settlement into spatially separated and morphologically distinct units (i.e. islets) should not be regarded as a prerequisite for pancreatic endocrine progenitor cells to differentiate into functionally active endocrine cells, although the endocrine cells in the cords and nests may perform significantly different functions in comparison to their related cells in the islets (see below). Two questions arise. Firstly, what is the function of the endocrine cells in the early epithelial cords and nests? Secondly, why do the majority of the endocrine cells locate in a separate compartment as soon as the epithelial cords and nests transform into a branching ductal system? The foetal endocrine cells in the epithelial cords and nests do not express the same levels of molecular markers present in adult pancreatic endocrine cells (Bernardo et al., 2008; Aye et al., 2010). For example, foetal human ␤-cells contain less insulin than expected based on corresponding mRNA data, suggesting that these cells rapidly secrete the peptide hormone after its translation and processing instead of storing it in secretory granules (Miettinen and Heikinheimo, 1992). Similarly, MAF (v-maf musculoaponeurotic fibrosarcoma oncogene family) proteins, which are involved in the acquisition of glucose-induced insulin secretion from secretory granules, are not expressed at high levels in foetal rat ␤-cells (AguayoMazzucato et al., 2011). In addition to the abovementioned molecular differences between foetal and adult endocrine cells, the endocrine cells in the cords and nests are also known to be less vascularised and innervated than their related cells in the islets. A stimulus secretioncoupling and metabolic-induced hormone release mechanism therefore appears less likely to be operational in the endocrine cells of the epithelial cords and nests. The early extrainsular endocrine cells therefore serve functions that are local, and not systemic in nature (Prasadan et al., 2002). This is in concordance with the observation that endogenous insulin production does not appear to be mandatory for maintaining normoglycaemia before birth (Duvillie et al., 1997). The two PC cleavage products of preproglucagon, GLP-1 and glucagon, are reported to play a role in islet tissue formation, maintenance, and renewal (Rall et al., 1973; Perfetti et al., 2000; Stoffers et al., 2000; Ling et al., 2001; Prasadan et al., 2002; Li et al., 2003; Stoffers, 2004; Gelling et al., 2009; Miller et al., 2009; Chung and Levine, 2010; Rojas et al., 2010). A similar trophic effect has been suspected for cleavage products of preproinsulin (Liu et al., 2011), although insulin itself appears to be a negative regulator of the ␤-cell mass (Duvillie et al., 2002), and insulin deficiency does not preclude pancreatic organogenesis and endocrine cell emergence (Duvillie et al., 1997). The specialised endocrine cells in the epithelium of the developing ductal system may thus preferentially support trophic rather than metabolic processes (i.e. driving ␤-cell differentiation and proliferation), and promote the formation of endocrine islets (Martin et al., 1984; Hole et al., 1988; Weinhaus et al., 2003; Navarro-Tableros et al., 2007; Miller et al., 2009; Rojas et al., 2010). Anatomical rearrangement of the majority of endocrine cells, and islet formation may become necessary when changing physiological conditions in the foetus necessitate that the developing pancreas maintain glucose homeostasis (Navarro-Tableros et al., 2007), or protection is needed for the endocrine cells from the exocrine juice secreted by the acini and the columnar epithelial cells lining the ducts.

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4. The cellular origin of the pancreatic endocrine component localizes to the primitive ducts The pancreatic endocrine and exocrine tissues influence the function of one another during development, and on into maturity (Leeson and Leeson, 1986; Bertelli and Bendayan, 2005). Their close functional relationship justifies their being considered as a single functional unit, regardless of their partly disparate morphological organizations. For example, the endocrine cells influence bicarbonate secretion by the ductal epithelium, as well as zymogen secretion by the acini either via paracrine release of islet hormones in the ducts, or endocrine delivery via an insulo-acinar portal bloodstream (Cruz et al., 1984; Park and Bendayan, 1992). In contrast, the exocrine acinar and duct cells are thought to influence the endocrine cells by secreting proteins (e.g. regeneratin proteins) which may subsequently be bound and endocytosed by the endocrine cells (Bertelli et al., 2001; Bertelli and Bendayan, 2005; Gurr, 2011). Serotonin- and cholecystokinin-containing endocrine cells also play a role in influencing pancreatic exocrine function. These cells are distinctly present in the epithelial lining of the proximal duodenal portion of the common pancreatic-bile duct and influence the tone of the muscular valve. This valve controls the flow of bile and pancreatic juice from the pancreatobiliary system into the duodenum (Park and Bendayan, 1992). It is noteworthy that disorders of the pancreatic endocrine tissue may also change properties of the pancreatic exocrine tissue and vice versa (Park and Bendayan, 1994). The close anatomical relationship between the pancreatic endocrine and exocrine tissues is readily apparent in the early outgrowths of the ventral and dorsal primordia (see above). At this time, all organ cells, regardless of their lineage, group in the epithelial cords and nests that invade the surrounding mesodermal mesenchyma (Fig. 1). The later separation of most of the pancreatic cells into exocrine and endocrine compartments results in an incomplete spatial segregation of the cells. The widely accepted, and often cited view of pancreatic islet formation is that islet progenitor cells initially detach and escape from the epithelial cords and nests early in development. They then disseminate in the surrounding primitive interstitial tissue and coalesce to form small cell clusters which later develop into islets (Fig. 2; Kim and Hebrok, 2001; Cleaver and Melton, 2005). This simplistic view of pancreatic islet formation appears to require modification (Bertelli and Bendayan, 2005; Jo et al., 2011; Merkwitz et al., 2013). In spite of the majority of endocrine cells locating in the islets later in development, a minority of these cells, so-called extra-insular endocrine cells, or ductal endocrine cells, continue to be intermingled with ductal and acinar elements through the growth and maturation phase of the organ (Fig. 3; Larsson et al., 1975; Redecker et al., 1992; Bertelli et al., 1994; Bouwens and Pipeleers, 1998; Lucini et al., 1998). These extra-insular endocrine cells, or ductal endocrine cells, distribute across the entire ductal system. At least in rodents, insulinand glucagon-immunoreactive cells are found to distribute in almost equal amounts across the pancreas, whereas somatostatin- and PPY-immunoreactive cells have been reported to accumulate in the proximal duodenal parts of the organ (Park and Bendayan, 1993). The ductal endocrine cells are not surrounded by a vascular network, and some reveal an apical access to the duct lumen and/or show close contact to adjacent duct cells (Bendayan, 1987; Bertelli et al., 2001). A location-specific paracrine function by transcellular and intercellular pathways seems probable. Indeed, most of the endocrine cells face the lumen of the duct, and

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Fig. 2. According to current concepts, founder cells of endocrine islets in the pancreas (cell highlighted in bold in A) segregate and migrate from the ductulo-acinar tree (B), after which they form small insular clusters (C) that are the founding structures of islets (D) at later stages of development.

contain apical domains rich in plasma membrane vesicles and secretory granules. They are interconnected to the epithelial duct or acinar cells by intercellular contacts, such as gap junctions (Bendayan, 1982). That pancreatic hormones have been reported to influence exocrine secretion of enzymes from the acini, and alkaline fluid rich in sodium bicarbonate from the ducts is not astonishing in this context (Youngs, 1972; Lee et al., 1990; Barreto et al., 2010). Interestingly, the percentage of ductal endocrine cells as a proportion of the total quantity of all endocrine cells appears to vary considerably between species (e.g. 1% in rodents, and 10 to 15% in humans and the normal adult pancreata of other species; Table 2 and Fig. 3; Rahier et al., 1981; Redecker et al., 1992; Bouwens and Pipeleers, 1998). Apart from single cells, ductal endocrine cells occur in pairs, or even small clusters in the adult pancreas (Fig. 3; Redecker et al., 1992). These clusters are predominantly found in peripheral ducts (i.e. intralobular collecting and intercalated ducts), are composed of ␤-cells, and are reported to bud from the duct epithelial lining, or to become separated from the ductal system by connective tissue emerging between them and the ducts. It has been suggested that these endocrine cell clusters represent a step in the transition from ductal islet precursor cells to islets, and should not be mistaken for, or subsumed to small islets (Kaihoh et al., 1986; Bertelli and Bendayan, 2005). Small islets are reported to offer higher therapeutic benefit than large islets when being transplanted to the portal vein in human and animal models (MacGregor et al., 2006; Lehmann et al., 2007). Non-vascularised endocrine cell clusters may contribute a high portion of dividing precursor cells to the islet transplants if they are present in the fraction of small islets. These cell clusters may also be more able to survive a hypoxic period than already vascularised true small islets.

5. Pancreatic endocrine islets continuously arise from cells of the expanding ducts As discussed previously, the development of the endocrine pancreatic islets is tightly associated with the development of the exocrine pancreatic tissue. Endocrine islets and

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Fig. 3. Light photomicrographs of paraffin sections (7 ␮m thick) of adult pancreata obtained from different species. Photomicrograpghs of immunohistochemical-labelled (all photomicrographs except D) and haematoxylin- and eosin-stained (D) sections are shown. In adult pancreata, the majority of endocrine cells locate in islets (stars), although some endocrine cells are seen in an extra-insular ductulo-acinar location (arrows). A,B: Only a few extra-insular, insulin-immunostained cells are found in either single or small cell clusters in murine (A) as compared to human (B) pancreata. C-F: The extra-insular, insulin-immunostained cells (arrows) are associated with ducts (circles in C,D) and acini (arrow heads in E,F). G,H: The extra-insular endocrine cells are identified by virtue of their immunostaining by antibodies directed against synaptophysin, a microvesicle marker (Redecker et al., 1991). Immunoreactive cells are shown in close vicinity to a duct (circle in G), as well as cells in division within an acinus (H). Scale bars as indicated.

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Table 2. Significant species differences between rodent, human, and bovine pancreata and pancreatic islet tissue. Species

rodent

Organ appearance

mesenteric

human/cattle compact

Endocrine Islets Islet type

Langerhans

Langerhans

Foetal Laguesse (ensured in cattle)

formation time

mainly postnatal

mid trimester

location size cell types

central » peripheral small - medium INS* ,GCG** ,PPY# , SST## ,GHRL@ separated in the islet core insulo-venous efferent » insulo-acinar portal substantial

mid and last trimester peripheral » central small-medium INS* ,GCG** ,PPY# , SST## ,GHRL@ intermixed in core-like structures insulo-acinar portal » insulo-venous efferent minor

distribution of ␤-cells islet vasculature

nerve supply

central large INS* the only cell type present insulo-venous efferent? substantial

Extra-insular Endocrine Tissue in Adult Pancreata ductal * ** # ## @

∼ 1%

10-15%

insulin glucagon, pancreatic polypeptide somatostatin ghrelin

exocrine tissue both originate in the cords and nests of epithelial cells that first arise from the two endodermal primordia. The islets form when the epithelial cords and nests are canaliculated, transform into primitive tubules and give rise to a tree-like system with ducts and acini at the sides and ends (O’Rahilly, 1983). The islets originate from progenitor or precursor cells present in the epithelial lining of the expanding ductal system. Islet generation starts within proximal ducts (i.e. in the very earliest ducts, and at distance from the lobule periphery), undergoes centrifugal evolution concomitant with the expanding ductal system, and ends in intercalated ducts at the outer parts of the lobules (Fig. 4). This leads to a temporal and spatial pattern of islet formation within the developing pancreas of humans (Polak et al., 2000; Jeon et al., 2009), mice, and cattle. Importantly, this process perhaps gives rise to true heterogeneity between islets within and between species (Table 2; Jorns et al., 1988; Steiner et al., 2010), rather than just differences between islets at different developmental steps in islet formation.

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Fig. 4. According to the model of duct-dependent heterogeneity between pancreatic islets, proximal duct cells give rise to endocrine islets that differ from the islets arising from duct cells at the intercalated duct/acinar interface. A: At the beginning of islet formation, proximal duct islets (e.g. foetal Laguesse islets in cattle; for detailed description see text) develop concomitant with intercalated duct islets (e.g. the classical Langerhans islets in cattle). B: At later developmental stages, proximal duct islets stop developing while intercalated duct islets continue to develop within the further expanding exocrine lobules. C: At the premature stage, proximal duct islets are found located in the connective tissue septae between lobules next to interlobular ducts, larger blood vessels and neuronal tissue elements. The intercalated duct islets, however, are found located within the exocrine lobules and are surrounded by acini. Inspired by Watanabe et al., 1999.

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Differences have been reported in the timing of pancreatic endocrine islet formation in a given species. Possible reasons for these discrepancies are: the lack of a generally agreed upon definition of the term “pancreatic islets”; the different course of pancreatic islet development in the tissue from the two primordia (see above); and the spatiotemporal dynamics of islet formation, which results in the presence of islets of different maturity within the developing pancreas. The defined time frame for the settlement of the majority of the pancreatic endocrine cells in islets as a separate compartment is species specific, and coincides with the degree of organ maturity at birth. In humans and cattle, the endocrine islets essentially develop during the mid and last foetal trimester (Goldman et al., 1982; Jeon et al., 2009; Meier et al., 2010), whereas in rodents they start to develop relatively late in gestation (mouse ed 18.5; rat ed 17.5), undergo substantial remodelling in the period immediately after birth, and only appear in definitive number at four to twelve weeks of postnatal age (Table 2; Bouwens and De Blay, 1996; Fowden and Hill, 2001; Miller et al., 2009; Herbach et al., 2011; Jo et al., 2011). This is in line with an overall lower stage of development and foetal maturity at birth in rodents (mice and rats) than in other species, such as cattle and humans (Reddy and Elliott, 1988). The most widely used islet assignment criteria are: complex cellular arrangement consisting of a certain number of mostly different endocrine cell types; a certain spatial extension; and a distinctive blood and nerve supply. According to these criteria, a pancreatic endocrine islet is defined as being composed of 100 to 5000 peptide hormone synthesizing and secreting cells filling a distinct space (more than 50 ␮m) with an underlying and supporting neuro-vascular bed (Huang et al., 2012). A thin layer of connective tissue fibres separate the islets incompletely from the surrounding pancreatic tissue. This layer is dense enough to constitute a connective tissue capsule which facilitates the isolation of islets, but appears permeable enough to allow a paracrine flow of secreted peptide hormones into the intercellular space (Redecker et al., 1992). Interestingly, the islet size distributions among various species appears to be similar, and independent of body size, which has lead to the suggestion that there is an intrinsic limit to endocrine islet size in the pancreas (Jo et al., 2011). In rodents, cattle, and humans, the islets develop mainly or exclusively from the epithelium at the sides and tips of the expanding ductal system, which itself develops from the two pancreatic primordia (see above). The pancreatic ductal system is an elaborate structure of interconnecting ducts consisting of a main and an irregular accessory duct, interlobular and intralobular collecting ducts, and intercalated ducts (Reichert and Rustgi, 2011). The pancreatic islets emanate from the different parts of the ductal system in a coordinated process that is hypothesized to include the following steps (Figs. 5 and 6): activation of pancreatic islet founder cells in the epithelium of the expanding ductal system; proliferation of the founder cells and formation of small clusters of peptide hormone-producing cells; protrusion or budding of these cell clusters into the inner luminal or outer interstitial surface of the ducts; bulging and subsequent isolation of the cell clusters from the epithelium of the expanding ductal system by connective tissue; infiltration of the cell clusters by a supporting neuro-vascular bed; and, finally, islet fusion or fission followed by rearrangement of the islet cells. Although the developmental steps necessary for the formation of pancreatic islets from islet precursor cells are easily hypothesized and understood, the anatomical evidence in

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Fig. 5. Light photomicrographs of haematoxylin- and eosin-stained paraffin sections (7 ␮m thick), as well as corresponding drawings of developing bovine Langerhans islets. Their development is characterized by intimate and long-lasting contact with the ductular tree. A: Langerhans islet tissue (framed) arising at the intercalated duct/acinar interface (arrow heads) and spreading into the space initially occupied by acinar cells (half ellipses). B: Islet tissue increasing in mass, elongating, bending and engulfing richly vascularised connective tissue (arrows) approaching the acinus from the outside. C: Langerhans islets enclosing a vascularised connective tissue cores almost entirely. The islets (framed) aggregate with islet tissue in the neighbourhood (stars) to form larger complexes. D: A historical drawing by Carly Seyfarth illustrating an islet (framed) at the intercalated duct/acinar interface in a human pancreas at a similar stage of development to that shown in C (Seyfarth, 1920).

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Fig. 6. Light photomicrographs of haematoxylin and eosin-stained paraffin sections (7 ␮m thick) of bovine pancreata (A-C) illustrating the development of Langerhans islets from side buds of larger intralobular ducts. A photomicrograph of a section of a human pancreas is also shown (D). A,B: A winding endocrine cell mass (star) is shown in clear relation to the side of a larger duct (circle). C: An endocrine cell mass is shown winding in a spiral around a central connective tissue core. D: A historical photomicrograph by Carly Seyfarth illustrating a human Langerhans islets (star) in development from a larger intralobular duct (circle; Seyfarth, 1920).

support of this hypothetical process remains astonishingly incomplete (Bouwens, 2004; Miller et al., 2009; Jo et al., 2011). The molecular regulators of this complex morphogenetic process are even less well elucidated (Miralles et al., 1998; Perez et al., 2005). The recruitment of the islet precursor cells in the epithelium of the ducts is likely to be under the control of various growth factors and ductal derived GLP-1 (Miralles et al., 1998; Tei et al., 2005). For example, disruption of transforming growth factor-␤ (TGF-␤) signalling at the receptor level in mice overexpressing a dominant-negative TGF-␤ type II receptor is reported to

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result in an increase in islet cells (Tulachan et al., 2007). In contrast, the development of islet cells in mice is reported to be delayed, and their separation from the ducts inhibited, when epidermal growth factor receptor signalling is disrupted (Miettinen et al., 2000). During the transition from the ducts to the islets, the corresponding cells cease to express a spectrum of cytoskeletal proteins indicative of their epithelial origin, and begin to express mesenchymal proteins, such as vimentin (Cole et al., 2009). Changes in the expression of cell-cell (e.g. EP-CAM, N-CAM and E-, N- and R-cadherins) and cell-extracellular matrix (e.g. integrins) adhesion molecules, as well as extracellular matrix degrading enzymes (e.g. matrix metalloproteinases) have also been observed (Esni et al., 1999; Cirulli et al., 2000; Nielsen and McNagny, 2009; Merkwitz et al., 2011). These changes may facilitate: separation of founder cells from the ducts cells; assembly and aggregation into pre-islet structures; and crossing of the ductal basement membrane at the epithelial-mesenchymal interface (Sjodin et al., 1995; Dahl et al., 1996; Cirulli et al., 1998; Esni et al., 1999). In addition, adequate levels and distributions of cell-cell adhesion molecules are required for the establishment of endocrine-cell responsiveness to glucose (Karaca, 2010; Santos-Silva et al., 2012). The switch from a duct-like cell type to a peptide hormone secreting islet cell type can be readily monitored by immunostaining the epithelial cell layer lining the ducts with antibodies directed against markers specific for simple epithelial cells (e.g. cytokeratins [CK] 8, 18 and 19 in humans and cattle, CK 20 in rodents), pancreatic endocrine cells in general (e.g. neurogenin 3, synaptophysin), or particular endocrine cells (e.g. insulin, glucagon, somatostatin and PPY) (Figs. 3 and 7; Bouwens et al., 1997; Redecker et al., 1991; Bouwens, 2004; Piper et al., 2004). It has not yet been adequately resolved whether the ductal endocrine cells that are present when islet formation begins contribute to the process of islet formation, or whether this process is entirely dependent on newly activated progenitor cells residing in the pancreatic ductal epithelium (Deltour et al., 1991; Bouwens, 2004). Pre-existing ductal endocrine cells appear less likely to contribute to the process of islet formation, as single or small clusters of these cells continue to exist in the ducts even though neogenesis of pancreatic islets ends later in life (Jo et al., 2011). Furthermore, ductal endocrine cells are smaller than their counterparts in the islets. As previously discussed, islet formation follows a temporal and spatial pattern. The location of the islets in the organ, as well as their size and architecture varies considerably depending on the time and place from which the islets arise (Fig. 4). If the islets develop early, and proximal in the ductal system (i.e. in ducts that still branch, expand and form lobules), they situate perilobularly, septally, and/or most centrally in the developing lobules alongside main and/or draining ducts. Such proximal duct islets are frequently encountered in rodents (Table 2 and Figs. 8 and 9; Murakami et al., 1997; Bouwens, 2004). These islets are impressive with regard to size and may be subsequently subdivided by fission (Bonner-Weir and Orci, 1982; Miller et al., 2009; Kilimnik et al., 2010). If the islets develop later at the periphery of the ductal system (where it consists of peripheral ducts), the islets situate in an intralobular location surrounded by acinar elements. Such peripheral duct islets represent the majority of islets encountered in cattle and humans (Table 2 and Fig. 8). It should be noted that septally-located islets with structural

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Fig. 7. Light photomicrographs of paraffin sections (7 ␮m thick) of pancreata from 38 to 57 cm crown rump length (CRL) bovine foetuses immunostained for cytokeration 8 (A-C) and E-cadherin (D,E). The Langerhans islet founding cells (star) are connected to the epithelial lining of the ducts and acini. They are characterized by round nuclei, small, pale amphophilic cytoplasms and negative immunostaining for the epithelial markers cytokeratin 8 (A-C) and E-cadherin (D,E). The lack of cytokeratin 8 and E-cadherin immunostaining enables differentiation of these endocrine cells from neighbouring cells in the ducts (circle) and acini. Molecular changes in the endocrine cells are indicative of an epithelial to mesenchymal transition which occurs during the transformation of duct-like cells into islet cells (Cole et al., 2009). Scale bars as indicated.

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Fig. 8. Light photomicrographs of haematoxylin- and eosin-stained paraffin sections (7 ␮m thick) of developing and adult pancreata obtained from different species. Islets are an outgrowth of the ductal system. They either protrude from large proximal ducts (circles in A-D) or grow out from terminal ends of the ductal system at the intercalated duct/acinar interface (dashed lines in E,F). Islets originating from the large proximal ducts remain in an interlobular septal location (C,D) close to proximal interlobular ducts and larger blood vessels (V in D). Those islets originating from peripheral ducts become located within lobules and are entirely surrounded by acinar elements (G-I). Thus, islets are heterogeneous with respect to their neighbouring tissue depending on their duct of origin. A-D: Representative pancreatic sections of 10 day postpartum (A-C) and adult mice (D). E-G: Pancreatic sections of last trimester bovine foetuses (E,F) and adult (G) cattle (CRL = crown rump length). H,I: Intralobular islets in adult human pancreatic sections. Scale bars as indicated.

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Fig. 9. Light photomicrographs of haematoxylin- and eosin-stained (all photomicrographs except F), as well as Sirius red collagen-stained (F) paraffin sections (7 ␮m thick) of developing (A-D) and adult (E,F) murine pancreata. In mice and other rodents, pancreatic islets arise alongside large ducts in the fibrous pancreatic tissue. A-D: Islets (stars) and lobules often arrange upon opposite sides of the duct from which they arise. As the exocrine tissue develops, the interlobular spaces between the islets and exocrine lobules greatly diminish. In addition, the islets become situated next to neighbouring lobules. This results in a perceived intralobular location of the islets at the end of development. Staining of the pancreatic tissue with Sirius red facilitates the identification of interlobular connective tissue surrounding the islets and larger ducts (circle) and blood vessels (V). Scale bars and embryonic days as indicated.

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relationships to lobule ducts and vessels are frequently reported in the foetal and newborn pancreata of these species (Murakami et al., 1997; Watanabe et al., 1999; Ruchelli, 2011). The peripheral duct islets are rather small and tend to fuse (Fig. 5; Bonner-Weir and Orci, 1982). As pancreatic islets are continuously generated from progenitor cells at the expanding portion of the ductal system, the location of newly formed islets changes progressively from more proximal ducts at distance from the lobule periphery to ducts in the outer parts of the lobules (from proximal, perilobular to peripheral, periacinar; Watanabe et al., 1999). Thus, there tends to be a pattern of centrifugal evolution in islet formation within a species (from larger ducts to smaller intralobularly-located ducts at the periphery of the lobules). The progressive shift in the location of the islet formation site from the organ centre to the organ periphery causes developmental as well as permanent heterogeneity of pancreatic islets within a species (Redecker et al., 1992; Watanabe et al., 1999; Polak et al., 2000; Jeon et al., 2009). This finding may be of pathophysiological significance for type 1 and 2 diabetes (Kilimnik et al., 2011). Heterogeneity of pancreatic islets between species may be caused by temporal shifts in the concomitant presence of two dynamic processes: pancreatic islet formation, as well as expansion and ramification of the ductal system within species. Irrespective of these two processes, it is noteworthy that the exocrine component still increases in mass when islet formation has already ceased in all of the considered species (around birth in humans and cattle, and after four to 12 weeks of age in mice; Watanabe et al., 1999; Ruchelli, 2011; Merkwitz et al., 2013). This results in the relative percentage of endocrine to connective tissue decreasing in the final phase of pancreatic development (Hisaoka et al., 1992; Watanabe et al., 1999). The interstitial space, abundant at the time islets are formed (during the mid to end trimester in humans and cattle and during the perinatal period in rodents), diminishes within and between the lobules to the extent that there is little connective tissue between lobules, and almost none within them at the end of organ development. This may create a false impression of an intralobular location of septal islets, particularly in rodents (Fig. 9). In this case, the lobules in the adult organ are closely apposed due to an acute-angled branching pattern (El-Gohary and Gittes, 2012).

6. Position-related differences in islet morphology and function As outlined above, islets develop from founder cells in the expanding and ramifying ductal epithelium by a multi-step process that includes separation of detached islet cell clusters from the residual epithelium. Once a critical cell mass is obtained by the isolated islet cell clusters, they initiate the development of a supporting vascular bed, and become connected to the pancreatic circulation. Islet blood flow is therefore established concomitant with the transformation of the cell clusters into islets (Brissova et al., 2006; Johansson et al., 2006; Gorczyca et al., 2010). Consequently, it is not surprising that the vasculature of a pancreatic islet is notably different depending on the location of the islet in relation to the pancreatic lobules (Table 2; Olsson and Carlsson, 2011). Interlobularly-located proximal duct islets seem to receive their blood from one to three afferent arterioles, and to deliver their blood after it has passed through the insular capillaries directly into a venous efferent vessel (termed collectively as

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Fig. 10. Light photomicrographs of haematoxylin- and eosin- stained paraffin sections (7 ␮m thick) of a type 1 neuro-insular complex (NIC) in a murine pancreas collected at day 10 postpartum. A typical murine pancreatic islet (star) is shown in intimate contact with ganglion nerve cell bodies (N, arrows). NIC of this kind are frequently encountered in rodent pancreata. Their role is largely unknown. The abundance of ganglion nerve cell bodies in close vicinity to islets in rodent pancreata suggests that the existence of NIC is not merely coincidental. These complexes may be involved in regulating islet function in these species. Note the larger duct (circle) located in close proximity of the NIC. Scale bars as indicated.

the insulo-venous efferent vessel system; Bonner-Weir and Orci, 1982; Murakami et al., 1993; Murakami et al., 1997). On the other hand, the vascular supply of the intralobularlylocated peripheral duct islets seems to be integrated into the blood supply of the exocrine tissue in the lobules. These islets empty the blood they receive into the capillaries among the adjacent exocrine cells after it has passed the insular capillary bed (termed collectively as the insulo-acinar portal vessel system; Bonner-Weir and Orci, 1982; Murakami et al., 1993; Murakami et al., 1997; Henderson and Daniel, 1979). Nerve innervation is also notably different depending on the location of an islet in relation to the lobules (Table 2). Interlobularly-located proximal duct islets are closely innervated by sympathetic nerve fibres probably ending on glucagon-immunoreactive ␣-cells, as well as parasympathic nerve fibres ending on both ␣-cells and insulin-immunoreactive ␤-cells (Rodriguez-Diaz et al., 2011). Intralobularly-located peripheral duct islets appear to be sparsely innervated by autonomic nerve fibres. Most of the fibres end here on blood vessels and not on endocrine cells (Rodriguez-Diaz et al., 2011). Therefore, endocrine cells in proximal duct islets appear to be directly influenced by autonomic nervous activity, whereas endocrine cells in peripheral duct islets appear to be indirectly influenced by autonomic nervous activity, via the regulation of the blood vessel tone and vascular blood flow. It is noteworthy that nerve cell bodies in juxtaposition to pancreatic islet tissue form type I neuro-insular complexes (NIC), which are observed in the septa of mammalian and nonmammalian vertebrate pancreata (Fujita, 1959; Jorns et al., 1988; Watanabe et al., 1989; Putti et al., 2000). Significantly, type I NICs appear to constitute a substantial proportion of rodent islets (Fig. 10; Morgan and Lobl, 1968; Serizawa et al., 1979; Persson-Sjogren et al., 2000; Persson-Sjogren, 2001). The described differences in islet innervation and vascularisation may influence stimulus-induced islet function and hormone secretion (Kelly et al., 2011; Barker et al.,

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2012). Autonomic nervous signals may play a lesser role in species with predominantly intralobularly-located peripheral duct islets (i.e. humans) than in species with predominantly interlobularly-located proximal duct islets (i.e. rodents; Rodriguez-Diaz et al., 2011). This could explain why euglycemia can occur after transplantation of denervated pancreata or islets in humans (Clark et al., 1989; Warnock et al., 1992; Cottrell, 1996; Hopt and Drognitz, 2000), whereas, in rodents, proper autonomic innervation is considered critical for an adequate islet cell response to blood glucose (Rodriguez-Diaz et al., 2011; Rodriguez-Diaz et al., 2012). On the other hand, islet hormones may have a stronger influence on acinar cell secretion when the blood reaching the acini first passes through the insular capillary bed (as seen in humans, primates, dogs, and horses) and does not reach the acini directly via vessels emerging form interlobular arteries (as often observed in rodents). Immunocytochemical and biochemical differences have been noted between the acini surrounding an islet (juxta-insular acini) and those at a distance from an islet (tele-insular acini; Kramer and Tan, 1968; Bendayan, 1985; Aughsteen and Kataoka, 1993). This finding supports the contention that there is a direct influence of islet hormones on neighbouring acinar tissue (e.g. uptake of glucose and amino acids; Leeson and Leeson, 1986).

7. Laguesse islets are a feature of foetal and neonatal pancreata in cattle, and probably in humans We have recently proposed that the duct based heterogeneity of pancreatic islets can, in its extreme, lead to two populations of entirely different pancreatic islets within one species (Merkwitz et al., 2013). Insulin secreting Laguesse islets and classical multi-hormonal Langerhans islets develop and co-exist in the foetal pancreas of cattle (Bonner-Weir and Like, 1980; Baltazar et al., 2000; Merkwitz et al., 2013). The classical Langerhans islets evolve continuously with the expansion of the ductal system at the intercalated ductulo-acinar interface (Figs. 4,5,8 and 11). They are composed of all the endocrine cells normally present in islets, possess rather sparse innervations, and are equipped with a core-to-mantle capillary system. This islet type resembles the aforementioned peripheral duct islets, locates within the exocrine lobules, is completely surrounded by acini, and persists beyond the neonatal period into later life (Figs. 8 and 11, Table 3). In contrast, Laguesse islets (which were observed by Laguesse (1896) in sheep more than a century ago) are few in number, and arise as large islets from proximal ducts (Figs. 4 and 11). They locate in the interlobular connective tissue septae in juxtaposition to larger ducts and blood vessels. Most noteworthy, these islets have a close and intimate spatial relationship with elements of the nervous system (intrapancreatic ganglia and nerve bundles) (Fig. 12; Baltazar et al., 2000; Merkwitz et al., 2013). It is reasonable to classify Laguesse islets according to their development and location as proximal duct islets. However, they show a number of features that render them unique in comparison to proximal duct islets of other species, rodents included. Firstly, Laguesse islets are transient in nature, restricted to foetal and early postnatal life. Secondly, they are composed of only one of the types of endocrine cells normally present in pancreatic islets, (i.e. columnar cytoplasm-rich ␤-cells). Finally, they are composed of epithelial trabeculae with a ribbon-like appearance containing gyriform and rosette-like arrangements (Fig. 11

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Fig. 11. Light photomicrographs showing Langerhans islets (star) and Laguesse islets (triangle) in paraffin sections (7 ␮m thick) of bovine pancreata at different stages of foetal bovine development, as indicated by the different crown rump lengths (CRL). The sections are either stained with haematoxylin and eosin (A,B,E), or immunostained with antibodies directed against simple epithelial cytokertain 8, (C,D), insulin (F,H), or glucagon (G,I). The formation of Langerhans and Laguesse islets in cattle exemplifies how islet biology, architecture and function may vary depending on the region of the ductal tree from which a pancreatic islet evolves. A: The classical Langerhans islets occur consistently within the exocrine area at the intercalated duct/acinar interface (arrowheads in A). B-D: The Laguesse islets develop early from side buds of more proximal ducts (arrows in B,C,D). E: At term, differences in location, size, architecture and cellular appearance between the two types of islets are striking. F,G: Differences in the occurrence of insulin (D,F) and glucagon (E,G) synthesizing cells between the two islet types. In classical bovine Langerhans islets (D,E, stars), glucagon/GLP-1immunostained cells occur alongside insulin-immunostained cells. In bovine Laguesse islets, (F,G, triangles) insulin-immunostained cells constitute the vast majority of the parenchymal cells. Scale bars as indicated.

and Table 3). The involution process of these islets is associated with parenchymal cell death, peliosis-like vascular ectasia, leucocytic infiltrates, as well as the initiation and progression of a fibrotic response (Fig. 13; Grossner, 1967; Titlbach et al., 1985; Merkwitz et al., 2013). Laguesse islets are accepted as being a distinctive feature of the foetal and neonatal pancreata of ruminants (Bonner-Weir and Like, 1980; Titlbach et al., 1985; Baltazar et al., 2000; Merkwitz et al., 2013), but it appears that they are not a peculiarity entirely unique to these mammals. Type 1 NICs with biological, structural, and functional similarities to Laguesse islets have been described in the older scientific literature concerning pancreas development in humans (Neubert, 1927; Van Campenhout, 1927; Liu and Potter, 1962; Jaffe et al., 1982), other vertebrate species, including monkeys (Girod et al., 1987; Liu

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Fig. 12. Light photomicrographs showing Laguesse islets (triangle) closely apposed to ganglion nerve cell bodies (N) and nerve fibres (double headed arrow) in paraffin sections (7 ␮m thick) of bovine pancreata at different stages of foetal development, as indicated by the different crown rump lengths (CRL). The sections are either stained with haematoxylin and eosin (all photomicrographs except E), or immunostained with antibodies directed against neurophysin (E). Laguesse islets are a morphologically and functionally distinct population of foetal interlobular proximal duct islets in cattle and other ruminant pancreata. A-E: The islets show an intimate morphological relationship with ganglion nerve cell bodies comparable to type 1 neuroinsular complexes in rodents (see Fig. 10). F,G: Nerve fibres entering the islets are accompanied by blood vessels (V). Scale bars as indicated.

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Fig. 13. Light photomicrographs of haematoxylin- and eosin- (A,B,C,F,G), as well as Sirius red-stained (D,E) paraffin sections (7 ␮m thick) of bovine pancreata during different phases of development (indicated by the different crown rump lengths; CRL) showing Laguesse islets (triangle) in different phases of involution. A-E: The involution program of the islets appears to be similar to the process of tissue remodelling which occurs during wound healing and inflammation. The physiologically normal, but pro-inflammatory remodelling comprises parenchymal cell death (A), peliosis-like vascular ectasia (B), invasion by immune cells (C), and deposition of fibrillary collagen (D,E). F,G: Dense lymphocytic infiltrates (LI) are often seen in close vicinity to the ganglion nerve cell bodies (N) associated with the islets. Scale bars as indicated.

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Table 3. Characteristics of Laguesse and Langerhans islets in foetal cattle* Laguesse islets

Langerhans islets

few; arising synchronously in a single episode from proximal ducts located in the interlobular connective tissue septae in close proximity to larger blood vessels and neuronal elements large in size characterised by epithelial trabeculae with gyriform and rosette-like appearances comprised almost entirely of cytoplasm-rich, weakly-immunoreactive ␤-cells with eccentric nuclei transitory, with perinatal involution

many; arising in several waves from ducts at the intercalated duct/acinar interface embedded within the exocrine lobules, surrounded by acini

*

small in size characterised by concentric, dense epithelial arrangements contain a significant portion of other islet hormone-bearing cells besides small-sized, strongly-immunoreactive ß-cells permanent, with perinatal persistence

Adapted from Merkwitz et al., 2013

et al., 1994), dogs, cats, rabbits (Fujita, 1959; Jorns et al., 1988; Watanabe et al., 1989), mice (Serizawa et al., 1979; Persson-Sjogren, 2001), rats (Morgan and Lobl, 1968), and fish (Putti et al., 2000). It has been suggested that, in some cases, foetal-type islets persist in newborn humans. Their suspected non-glucose dependent release of insulin may contribute to persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI) (Fig. 14; Shermeta et al., 1980; Titlbach et al., 1985; Kassem et al., 2000; Sempoux et al., 2011). The same scenario may apply when foetal type islets reappear after Roux-en-Y gastric bypass surgery (Service et al., 2005; Rumilla et al., 2009; Cui et al., 2011; Rabiee et al., 2011). On the other hand, a misdirected involution of foetal type islets, and nearby neuronal elements around birth (when the islet tissue reorganizes on a larger scale in mammalian pancreata; Scaglia et al., 1997; Kassem et al., 2000; Trudeau et al., 2000; O’Brien et al., 2002; Bonner-Weir et al., 2010) may promote postnatal insulitis, and finally lead to type 1 diabetes (T1D, Fig. 14; Kassem et al., 2000; Mathis et al., 2001; Winer et al., 2001; Turley et al., 2003; Winer et al., 2003; Persson-Sjogren et al., 2005; Razavi et al., 2006; Tsui et al., 2008; Katz and Janssen, 2011; Tsui et al., 2011). We recently showed that the involution of Laguesse islets in cattle possessed a wound healing/inflammatory signature (Merkwitz et al., 2013). We propose that debris from ␤cells and nerve cell bodies initiate a mild inflammatory process. We speculate that the debris is phagocytosed, processed by myeloid cells, and presented as bioactive fragments to lymphocytes, which are either diffusely dispersed throughout the foetal pancreas, or domiciled in lymphatic follicles or draining lymph nodes (Jansen et al., 1993; HomoDelarche and Drexhage, 2004; Ruchelli, 2011). We propose that this process normally leads to an auto-protective immune and tissue response comprising high matrix metalloproteinase activity, increased cytokine levels, and fibrillar collagen deposits. A pathological autoreactive response could potentially result, if this process is subverted.

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Fig. 14. The pancreatic ␤-cell mass is remodelled during the perinatal period. The morphological reorganization appears to proceed within narrow biological limits. We and others (Shermeta et al., 1980; Mathis et al., 2001) suggest that insufficient or excessive levels of ␤-cell mass remodelling may lead to hyperinsulinaemic, hypoglycaemic and hypoinsulinaemic hyperglycaemic diseases.

A goal of our future studies concerning bovine Laguesse islets will be to elucidate their specific gene profile to guide us in the identification of such foetal type islets in non-ruminant species, especially mice and humans.

8. Comments on studies involving the experimental manipulation of pancreatic islet formation During development, the major mechanism causing the increase in islet mass is neogenesis from an undifferentiated ductal progenitor or precursor cell, and not self-renewal of pre-existing differentiated islet cells (Deltour et al., 1991; Bouwens and Rooman, 2005). After the islet mass is formed and complete, islet cells renew under physiological conditions,

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primarily by replication. This mechanistic switch is clearly evident in rodents (Georgia and Bhushan, 2004; Solar et al., 2009). However, if the islet cell mass of the pancreas is reduced by pancreatectomy or pharmacological methods, or the flow of the pancreatic juice to the duodenum is inhibited by duct ligation, ␤-cell neogenic events are initiated that are similar to those occurring during pancreatic development (Bonner-Weir et al., 1993; Wang et al., 1995; Duvillie et al., 2002; Inada et al., 2008; Demeterco et al., 2009; Pan et al., 2013). Facultative reactivation of numerous transcription factors (Sox9, Hnf1␤, NGN-3) in the injured organ stimulates ␤-cell neogenesis and increases the islet mass (Xu et al., 2008). It is yet unclear which particular cell type (activated progenitor, precursor, transdifferentiated acinar, or duct cell) is initially involved in the process (Xu et al., 2008; Pan et al., 2013). As outlined above and stated in many of the cited studies, pancreatic islets differ considerably within and between species with regard to developmental time frame, ductal origin, relation to surrounding stromal and exocrine tissue, islet architecture, cellular composition, and blood and nerve supply. In particular, these differences need to be considered when extrapolating results obtained from rodent studies to humans (especially if the results are claimed to have translational potential for human islet or ␤-cell replacement therapies; Steiner et al., 2010). As discussed in this review, foetal/neonatal type islets resembling Laguesse islets of ruminants are possibly present in the foetal and neonatal pancreata of humans and other species. Pathological persistence of such islets therefore needs to be considered before focal adenoma-like lesions are diagnosed as the cause for an observed clinical picture of PHHI, since insulin release from these islets may also be poorly responsive to glucose (McMillen and Robinson, 2005). Pathological persistence of foetal/neonatal-type islets beyond the neonatal period, or the recurrence of these islets after bariatric Roux-en-Y gastric bypass surgery should be considered as a potential source of hyperinsulinaemic events in infancy and adulthood. It has been suggested that the presence or absence of fibrosis, or compression of surrounding organ tissue and cytological appearance of the islet cells could be used to distinguish islet cell hyperplasia from islet adenoma-like lesions. Our own and other findings suggest that Laguesse-type islets and hyperplastic lesions may follow comparable regressive changes when they begin to dissipate (Jaffe et al., 1982; Titlbach et al., 1985; Merkwitz et al., 2013). We are therefore in full agreement with Ruchelli (2011), who states that the unique features of the endocrine tissue of foetal/neonatal pancreata has to be considered when the possible cause of either presumed or clinically-established hyperinsulinism is traced. The well-differentiated ribbon-like appearance of bovine Laguesse islets not only resembles focal hyperplasic lesions in PHHI or after Roux-en-Y gastric bypass surgery, but also microadenomatosis (Anlauf et al., 2006) and insulinoma (Sempoux et al., 2003) in humans, and the so-called “mega islets” in non-obese diabetic mouse models for T1D (Rosmalen et al., 2000; Rosmalen et al., 2001; Duvillie et al., 2002). The hyperplastic islet tissue in most of these lesions consists of cytoplasm-rich ␤-cells with a faint, diffuse, homogenous insulin immunostaining pattern (Sempoux et al., 2003; Sempoux et al., 2011). It is likely that their immunostaining properties reflect an autonomous insulin secretory behaviour (Titlbach et al., 1985; Sempoux et al., 2011). The existence of morphologically-distinct populations of islets in situ suggests that pancreatic digests prepared for islet replacement therapy in humans are composed of a

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heterogeneous mixture of islets. Indeed, when the characteristics of rat islet populations in situ are compared to isolated islets, it was found that the widely reported negative correlation between insulin content and islet size in vitro (MacGregor et al., 2006; Lehmann et al., 2007; Fujita et al., 2011; Huang et al., 2012) is mirrored by a comparably lower cell density and cellular insulin content of large islets in vivo (Huang et al., 2011). Further studies are needed to examine whether procedures in which only small, or peripheral duct islets are transplanted are indeed superior to procedures in which all harvested islets are transplanted regardless of their sizes. Studies are also needed to determine whether the apparent beneficial transplantation properties of small islets are a consequence of their enhanced low oxygen resistance, revascularisation capability, proliferation capacity, and/or other additional beneficial properties (Lehmann et al., 2007). The two most prevalent methods for isolating islets from pancreatic tissue differ primarily in the way digestive enzymes are introduced to the pancreatic tissue surrounding the islets (van Suylichem et al., 1992; Carter et al., 2009). In the first method, the pancreas is excised and cut into small pieces which are then subjected to enzymatic digestion at 37 ◦ C under mechanical stirring or shaking. In the second method, a cold enzyme solution is first injected into the proximal ductal system before the pancreas is incubated as a whole or in pieces at 37 ◦ C under mechanical agitation. It would be interesting to determine whether differences exist between the ratios of small peripheral duct islets to large proximal duct islets in preparations resulting from the two methods. The isolation and in vitro expansion of islet progenitor or precursor cells from pancreatic and bilary ducts is a much sought after goal because of the potential benefit of these cells for in vitro neogenesis of transplantable ␤-cells (Bonner-Weir et al., 2000; Oshima et al., 2007; Sugiyama et al., 2007; Inada et al., 2008; Nagaya et al., 2009). In this context, one should consider that pancreatic duct tissue or cells in relation to their respective origin within the ductal tree may give rise to two different types of islets. Indeed, as we have discussed above, there exist location- and size-related functional differences among pancreatic islets within one species.

9. Summary In this review, we focus on the close developmental relationship between the expansion of the ductal system and the occurrence of endocrine islets. We discuss the variations in the microarchitecture of the islets, as well as the vasculature and nerve fibres associated with these structures within and between species. We speculate that the variations in islet microarchitecture are dependent on the timing of ductal outgrowth and the location of the duct cells that give rise to the islets. Furthermore, we note that in the extreme, these two processes lead to either large, proximal, more rodent type islets, or smaller, peripheral, more bovine/human type islets (i.e. islets mainly located in the septae or surrounded by acini; draining into the venous efferent vessel or an insulo-acinar portal system; being under direct or indirect neural input, the latter by innervations of the supporting blood vessels) (Table 3). We report observations indicating that foetal Laguesse islets, which constitute a foetal variant of large proximal duct islets in ruminants, are characteristic, but not exclusive to ruminants. The possibility that pathological persistence or reappearance of foetal type islets

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may be a causative factor for hyperinsulinaemic events in infancy and adulthood, as well as in insulitis is considered. Collectively, we suggest that structural and functional heterogeneity between pancreatic islets is based on the different regions of the ductal tree from which the particular islets in a species arise. Clearly, more studies are needed concerning this islet heterogeneity. Acknowledgements We greatly enjoy our ongoing collaborations and discussions concerning the development of the pancreas with colleagues from institutes in Leipzig and abroad. Our particular thanks go in alphabetical order to Ingo Bechmann, Jan Böttger, Rolf Gebhardt, Martin Gericke, Sonja Kallendrusch, Franz-Josef Kaup, Ingrid and Nora Klöting, Stine Kremzow, Madlen Matz-Soja, Michiharu Sakurai, Torsten Schöneberg, and Hanno Steinke. We thank the publishers of Journal of Anatomy for allowing the use of previously published data as part of this review. Paul Lochhead is supported by a fellowship from the Chief Scientist Office of the Scottish Government. Jaroslawna Meister is member of the IFB MD program at the University of Leipzig. References Abraham EJ, Leech CA, Lin JC, Zulewski H, Habener JF. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology 2002;143:3152–61. Aguayo-Mazzucato C, Koh A, El Khattabi I, Li WC, Toschi E, Jermendy A, Juhl K, Mao K, Weir GC, Sharma A, Bonner-Weir S. Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells. Diabetologia 2011;54:583–93. Anlauf M, Schlenger R, Perren A, Bauersfeld J, Koch CA, Dralle H, Raffel A, Knoefel WT, Weihe E, Ruszniewski P, Couvelard A, Komminoth P, Heitz PU, Kloppel G. Microadenomatosis of the endocrine pancreas in patients with and without the multiple endocrine neoplasia type 1 syndrome. The American journal of surgical pathology 2006;30:560–74. Aughsteen AA, Kataoka K. Morphometric studies on the juxta-insular and tele-insular acinar cells of the pancreas in normal and streptozotocin-induced diabetic rats. Journal of electron microscopy 1993;42:79–87. Aye T, Toschi E, Sharma A, Sgroi D, Bonner-Weir S. Identification of markers for newly formed beta-cells in the perinatal period: a time of recognized beta-cell immaturity. J Histochem Cytochem 2010;58:369–76. Baetens D, Malaisse-Lagae F, Perrelet A, Orci L. Endocrine pancreas: three-dimensional reconstruction shows two types of islets of langerhans. Science 1979;206:1323–5. Baltazar ET, Kitamura N, Hondo E, Narreto EC, Yamada J. Galanin-like immunoreactive endocrine cells in bovine pancreas. J Anat 2000;196:285–91. Barker CJ, Leibiger IB, Berggren PO. The pancreatic islet as a signaling hub. Adv Biol Regul 2013;53:156–63. Barreto SG, Carati CJ, Toouli J, Saccone GT. The islet-acinar axis of the pancreas: more than just insulin. Am J Physiol Gastrointest Liver Physiol 2010;299:G10–22. Bendayan M. Contacts between endocrine and exocrine cells in the pancreas. Cell Tissue Res 1982;222:227–30. Bendayan M. Morphometrical and immunocytochemical characterization of peri-insular and tele-insular acinar cells in the rat pancreas. Eur J Cell Biol 1985;36:263–8. Bendayan M. Presence of endocrine cells in pancreatic ducts. Pancreas 1987;2:393–7. Bernardo AS, Hay CW, Docherty K. Pancreatic transcription factors and their role in the birth, life and survival of the pancreatic beta cell. Mol Cell Endocrinol 2008;294:1–9. Bertelli E, Bendayan M. Association between endocrine pancreas and ductal system. More than an epiphenomenon of endocrine differentiation and development? J Histochem Cytochem 2005;53:1071–86. Bertelli E, Regoli M, Bastianini A. Endocrine tissue associated with the pancreatic ductal system: a light and electron microscopic study of the adult rat pancreas with special reference to a new endocrine arrangement. Anat Rec 1994;239:371–8.

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