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Cell Biology International (2011) 35, 1065–1078 (Printed in Great Britain)
Review article
Islet organogenesis, angiogenesis and innervation
Marlon E. Cerf1
Diabetes Discovery Platform, South African Medical Research Council, PO Box 19070, Tygerberg, 7505 Cape Town, South Africa


The pancreas is characterized by a major component, an exocrine and ductal system involved in digestion, and a minor component, the endocrine islets represented by islet micro-organs that tightly regulate glucose homoeostasis. Pancreatic organogenesis is strictly co-ordinated by transcription factors that are expressed sequentially to yield functional islets capable of maintaining glucose homoeostasis. Angiogenesis and innervation complete islet development, equipping islets to respond to metabolic demands. Proper regulation of this triad of processes during development is critical for establishing functional islets.


Key words: β-cells, differentiation, endocrine, pancreatic development, transcription factor

Abbreviations: Btg1, B-cell translocation gene-1, BMP, bone morphogenetic protein, CTGF, connective tissue growth factor, Dnmt1, DNA methyltransferase 1, FGF10, fibroblast growth factor-10, FoxA1, forkhead box protein A1, GDNF, glial-derived neurotrophic factor, Hdac1, histone deacetylase 1, Hh, hedgehog, HGF, hepatocyte growth factor, Hhex, haemopoietically expressed homeobox, Hnf6, hepatic nuclear factor 6, MafB, musculo-aponeurotic fibrosarcoma B, MSC, mesenchymal cells, NCAM, neural cell adhesion molecule, NeuroD1, neurogenic differentiation 1, NGF, nerve growth factor, Ngn3, neurogenin 3, Nkx6.1, Nk homeobox 6.1, p75 NTR, p75 neurotrophin receptor, Pax4, paired box 4, Pdx1, pancreatic and duodenal homeobox 1, Prox1, prospero homeobox protein 1, Ptf1a, pancreas transcription factor 1a, Reep5, receptor accessory protein 5, Rfx, regulatory factor X, Sox17, sex-determining region on Y box 17, Stxbp6, syntaxin binding protein 6, TGFβ, transforming growth factor β, TH, tyrosine hydroxylase, TrkA, tropomyosine kinase-related receptor A, VEGFA, vascular endothelial growth factor A, VEGFR, VEGF receptor

1To whom correspondence should be addressed (email marlon.cerf@mrc.ac.za).


1. Introduction

Embryogenesis refers to the formation of anlagen before specific factors are activated to drive organogenesis. The pancreas shares ancestry with the stomach, intestine and liver, which are digestive in nature and characteristic of the exocrine portion, while the liver is also associated with the endocrine pancreas due to its role in regulating glucose homoeostasis. Pancreatic organogenesis involves the budding of the pancreas that progresses to the differentiation of exocrine, ductal and endocrine constituents of the pancreas. The exocrine pancreas, defined by acinar cells, is closely associated with a ductal system, consisting of epithelial duct cells, and the endocrine unit is arranged as islets. In humans, the first pancreatic endocrine cells are detected around weeks 7–8 of development (Polak et al., 2000) with islet formation initiated at 12 weeks, vascularization from week 16 and innervation at mid-gestation (Fetita et al., 2006).

The exocrine pancreas produces and secretes several digestive enzymes. The collection and transport of these enzymes are undertaken by an intricate ductal network into the rostral duodenum (Wescott et al., 2009; Zhang et al., 2009). Although the acinar and duct cells may initially share some common transcription factors early in pancreatic development, these distinct cell types possess their own set of transcription factors specific for their differentiation and maturation. Both early exocrine and endocrine pancreas development are dependent on proper spatial and temporal expression of the key transcription factor Pdx1 (pancreatic and duodenal homeobox 1) (Wescott et al., 2009). The endocrine pancreas secretes several hormones that regulate glucose homoeostasis. This review focuses on transcription factors that play key roles in endocrine pancreas development and differentiation. Further, angiogenesis and innervation during pancreatic development are briefly discussed. Ultimately islets are formed to establish viable and functional endocrine units with distinct subpopulations of α-, β-, δ-, PP and ε-cells.

2. The pancreatic exocrine and ductal system

The transcription factors Ptf1a (pancreas transcription factor 1a) and BHLHA15 (basic helix–loop–helix family, member A15; Mist1) are required to determine the exocrine fate, but are not sufficient (Bonal and Herrera, 2008). The novel gene, exdpf (exocrine differentiation and proliferation factor), is specifically employed by zebrafish exocrine progenitor cells to promote cell differentiation and proliferation (Jiang et al., 2008). Wnt/β-catenin and Notch signalling pathways participate in the expansion and differentiation of exocrine progenitor cells (Bonal and Herrera, 2008). Genetic studies reveal that Hdac1 (histone deacetylase 1) is required for the establishment of hepatic and exocrine pancreatic cell fates within the foregut, which occurs at the expense of the tissue forming the alimentary canal, suggesting a model in which an epigenetic enzyme(s) mediates a fate switch at the organ level (Noel et al., 2008). In the adult, maintenance of the acinar cell mass relies on the activity of protective genes such as srf (serum response factor), while hnf6 (hepatic nuclear factor 6) is required for the ductal lineage (Bonal and Herrera, 2008).

In the neonatal rat pancreas, Ndrg4 was found in pancreatic duct cells and developing acinar tissue, but was absent from endocrine cells within islets (Wang and Hill, 2009). The expression of both n-Myc and Ndrg4 in pancreatic duct cells demonstrates further similarities in cell differentiation pathways in the pancreas and CNS (central nervous system) (Wang and Hill, 2009). Hnf6 is essential for both ductal and endocrine development (Zhang et al., 2009), likely functions upstream of another transcription factor, Prox1 (prospero homeobox protein 1), and may regulate duct morphogenesis partially through Prox1 (Zhang et al., 2009). Sox17 (sex-determining region on Y box 17) overexpression in the pancreas at e12.5, when it is not normally expressed, is sufficient to promote the ductal fate, at the expense of endocrine cells (Spence et al., 2009). This suggests that Sox17 acts at the top of a hierarchy and can activate a transcriptional programme resulting in the ductal fate (Spence et al., 2009).

Trans- or de-differentiation of mature cells or re-activation of the embryonic organogenic machinery has been demonstrated in the regenerating pancreas by acinar cell de-differentiation to the duct-cell-like phenotype, accompanied by the re-activation of the Notch signalling pathway after cerulean-induced pancreatitis (Jensen et al., 2005; Siveke et al., 2008). These studies demonstrate the close association of the exocrine pancreas and ductal system during development.

3. Embryogenesis and organogenesis of the endocrine pancreas

Gastrulation in the developing embryo [e7.5 (embryonic day 7.5) in mice] results in the formation of three germ layers, namely the ectoderm, the endoderm and the mesoderm (Mfopou et al., 2010). Pancreas development can therefore be defined in simple terms as a series of bifurcating lineage decisions: endoderm against mesoderm and ectoderm; pancreas against duodenum; exocrine against endocrine; and β-cell against other hormone-positive cell types (Guney and Gannon, 2009). Specifically, the pancreas develops from a region of the foregut endoderm located posterior to the developing liver and anterior to the duodenum (Guney and Gannon, 2009). During embryogenesis, the anlagen of the pancreas arise from both dorsal and ventral evaginations of the foregut that later fuse to form a single organ (Hayata et al., 2009). The ventral foregut endoderm of the mammalian embryo is arranged as simple epithelium of several hundred cells, yet it gives rise to a spectrum of organs and structures, including the lungs, thyroid, liver and hepatobiliary system (intrahepatic bile ducts, extrahepatic bile ducts, common duct, gall bladder and cystic duct) and ventral portions of the duodenum, stomach and pancreas (Spence et al., 2009). The biliary system is derived from the same region of the ventral foregut as the liver and pancreas (Spence et al., 2009). Sox17 is involved in early biliary development; it appears that Sox17, Hhex (haemopoietically expressed homeobox) and Hnf6 form a transcriptional network that regulates development of the biliary system (Spence et al., 2009). The ventral pancreas also arises from the same region of the ventral foregut as the hepatic and biliary system and loss of the Notch effector, Hes1, results in gall bladder agenesis and ectopic pancreatic tissue in the common duct (Spence et al., 2009).

Pancreatic buds grow, fuse and branch to eventually form the definitive pancreas (Hayata et al., 2009) by undergoing three developmental transitions characterized by extensive cell proliferation and differentiation (Jorgensen et al., 2007). Specific growth and differentiation factors released by adjacent tissues control sets of transcription factor expression, resulting in the patterning of the ventral and dorsal prepancreatic endoderm (Mfopou et al., 2010). The initial interactions usually confer competence to respond to additional inductive signals that establish organ determination and specification at particular time points, referred to as ‘competence windows’ (Mfopou et al., 2010).

Pancreatic epithelium responds in a cell autonomous manner to threshold levels of Ngn3 (neurogenin 3) that determine the number of cells shunted into the endocrine pathway (Johansson et al., 2007). In the mouse, the pancreas originates approx. e9.5 from dorsal and ventral evaginations of the gut tube endoderm (Villasenor et al., 2008). These pancreatic buds invade and grow into the visceral mesoderm (Villasenor et al., 2008). Shortly after e10.5, the gut tube rotates, resulting in the fusion of the dorsal and ventral pancreatic buds and the formation of a single organ (Villasenor et al., 2008). The pancreatic epithelium then branches, initially extending short finger-like lobules into the mesoderm (Villasenor et al., 2008). From e12.5 to birth, branching expands, creating a three-dimensional organ that grows dramatically throughout gestation (Villasenor et al., 2008). During this time, the pancreatic epithelium undergoes a number of dynamic cellular changes, giving rise to a tree-like, tubular epithelial network (Villasenor et al., 2008). Along the trunk of the developing pancreatic tree, endocrine progenitor cells delaminate as individual cells from the endodermal epithelium (Villasenor et al., 2008). These progenitor cells migrate and coalesce into small islet-like clusters, which progressively join and proliferate into larger endocrine aggregates (Villasenor et al., 2008). Substantial cell proliferation and differentiation culminates in the formation of the exocrine pancreas (consisting of acinar tissue and ductal epithelium) and endocrine pancreas (consisting of glucagon-secreting α-cells, insulin-secreting β-cells, somatostatin-secreting δ-cells, pancreatic polypeptide-secreting PP cells) (Jorgensen et al., 2007) and the ghrelin-secreting ε-cells. This establishes the digestive exocrine pancreas and functional islets that are capable of regulating glucose homoeostasis.

4. Signalling pathways involved in pancreatic development

Several signalling pathways are involved in pancreatic development. These include BMP (bone morphogenetic protein), Hh (Hedgehog), Wnt, Notch, retinoid, VEGF (vascular endothelial growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), TGFβ (transforming growth factor β) and PI3K (phosphoinositide 3-kinase) signal transductions (Champeris and Jones, 2010; Mfopou et al., 2010).

BMP signalling to the mesenchyme is crucial for the development of the pancreas (Ahnfelt-Ronne et al., 2010). BMP signalling, determined by pSmad1,5,8 immunoreactivity, is restricted to the mesenchyme during early pancreas development (Ahnfelt-Ronne et al., 2010). Inhibition of BMP signalling in vivo by Noggin results in reduced epithelial branching and increased endocrine differentiation that coincides with a severe failure of vascular remodelling and mesenchymal morphogenesis (Ahnfelt-Ronne et al., 2010). The effect of Noggin on endocrine differentiation can be mimicked by cell autonomous BMP signalling inhibition in the mouse pancreas mesenchyme using lentiviral transduction in vitro (Ahnfelt-Ronne et al., 2010).

The Hh signalling pathway plays a critical role in pancreas development and function (Cervantes et al., 2010). Loss- and gain-of-function studies in mice have revealed that deregulation of Hh activity affects pancreas morphogenesis and function (Lau et al., 2006). Elimination of cilia in the presence of GLI2ΔN in mice results in overt Hh activation in pancreatic epithelium and, consequently, impaired pancreas formation (Cervantes et al., 2010).

Primary cilia play a key role in regulating Hh signalling during pancreas formation; excessive Hh activation results in unique phenotypes in the pancreas, therefore there may be a potential role for Hh signalling in modulating the differentiated state of pancreatic cells (Cervantes et al., 2010). These cells expressed pancreatic progenitor markers, such as Hes1, FoxA2 (forkhead box protein A2) and Sox9, suggesting that activation of Hh impairs the ability of pancreatic cells to maintain a differentiated state (Cervantes et al., 2010).

5. Transcription factor regulation of islet development

Transcription factors actively regulate pancreatic development throughout organogenesis, differentiation and maturation. Table 1 lists some of the key transcription factors involved in pancreatic organogenesis and islet differentiation, focusing on those critical factors that are involved in establishing functional β-cells and are discussed in this review. It is important to note that these transcription factors can be involved at multiple levels of pancreatic development, i.e. during organogenesis, differentiation and maintaining the functional β-cell, e.g. Pdx1. For the purposes of this review, transcription factors involved in organogenesis and differentiation are discussed.


Table 1 Critical transcription factors regulating pancreatic organogenesis and islet differentiation

Pancreatic transcription factor
FoxA1
Hdac1
Hex1
Hnf6
Isl1
MafA
MafB
Ndrg4A2
NeuroD1
Ngn3
Nkx6.1
Pax4
Pax6
Pdx1
Prox1
Rfx3
Rfx6
Sox9



Organogenesis of the pancreas is spatio-temporally controlled by the sequential activation of a specific cascade of transcription factors (Prasadan et al., 2010). Analysis of the transcriptomes of precursor cells present at different stages of pancreas development is expected to further facilitate a definition of the genetic cascades essential for endocrine and exocrine differentiation (Hoffman et al., 2008). Both extrinsic and intrinsic factors regulate the differentiation and proliferation of endocrine cells in order to generate the normal proportions of each of the cell types within the pancreas (Guney and Gannon, 2009). The dynamic process of pancreatic development is controlled by extrinsic signals from the adjacent tissues and intrinsic transcription factors (Jiang et al., 2008). A network of intrinsic transcription factors that act in a cascade fashion to initiate and maintain cell-specific gene expression patterns determines the ultimate lineage-specific cell fate (Jiang et al., 2008).

Pancreatic organogenesis is orchestrated by interactions between the epithelium and the mesenchyme (Ahnfelt-Ronne et al., 2010) and depends on the precise execution of discrete gene-expression cascades and the acquisition of stable cell identities from multi-potent progenitor cells (Anderson et al., 2009). Complex multi-organ systems, such as the digestive system, in which the liver, exocrine and endocrine pancreas arise from the foregut endoderm, demand tightly co-ordinated steps of patterning and morphogenesis to allow formation of each organ (Noel et al., 2008). The development of a multi-organ structure (liver, lung and pancreas) requires the temporally and spatially co-ordinated regulation of gene expression (Noel et al., 2008). Neighbouring groups of cells, which initially share a common gene-expression programme, will adopt different fates by expressing different sets of genes (Noel et al., 2008). This is realized by actively regulated initiation and termination of transcription, which in turn depends on the presence of specific activating and repressing transcription factors, and importantly, on their ability to access regulatory gene elements (Noel et al., 2008).

Several transcription factors are involved in endocrine pancreas organogenesis. Sox17 is necessary for definitive endoderm formation in many vertebrate species including Xenopus, zebrafish and mouse (Spence et al., 2009). Sox17 is broadly expressed in foregut progenitor cells and is progressively down-regulated in cells as they become lineage restricted (Spence et al., 2009). Consistent with this, misexpression of Sox17 throughout the dorsal Pdx1 domain is sufficient to induce ectopic expression of Hhex in the duodenum and dorsal pancreas at e10.5 (Spence et al., 2009). Moreover, Sox17 misexpression is sufficient to induce the biliary/ductal molecular programme resulting in Hnf6 expression in ectopic ductal structures throughout the Pdx1 expression domain (Spence et al., 2009). These results suggest that Sox17 might act as a master regulator of the biliary/ductal lineage upstream of Hhex and is sufficient for diverting other cell types into the biliary lineage (Spence et al., 2009).

Down-regulation of Sox17 in Pdx1-expressing cells is required for pancreas development (Spence et al., 2009). One transcription factor, for example Sox17, might act in combination with others factors like Hex1 (hexokinase 1) and Pdx1 to specify different organ lineages from a common pool of progenitor cells (Spence et al., 2009). The proper expression of Pdx1 is essential for pancreatic exocrine and endocrine cell development and the maintenance of adult β-cell function (Fernandez-Zapico et al., 2009). This illustrates that specific transcription factors need to be up-regulated during defined critical developmental windows in the pancreas, while others are down-regulated and that tight co-ordination of transcription factor regulation during pancreatic development is required. This is critical for normal islet development.

During pancreas development, the first insulin cells express MafB (musculo-aponeurotic fibrosarcoma B) and then switch to MafA instead, after Nkx6.1 (Nk homeobox 6.1) and Pdx1 induction, like mature β-cells (Bonal and Herrera, 2008). Klf11 (Kruppel-like factor-11), which has a role in pancreatic expression, was enriched in the nuclei of human and mouse islet cell expression and at lower levels in acinar cells (Fernandez-Zapico et al., 2009). The misexpression of Pdx1 in the endocrine pancreas results in β-cell inactivity (Fernandez-Zapico et al., 2009).

MafA controls many genes first regulated by MafB in developing β-cells (Artner et al., 2010). Although other closely related transcription factors are synthesized within the pancreas [e.g. Pax4 (paired box 4)/Pax6, Nkx6.1/Nkx6.2 and FoxA1/FoxA2], none seem to act as specifically and in such a co-ordinated manner to impact islet cell function as MafA and MafB (Artner et al., 2010). MafB appears to be a more potent regulator of β-cell development than MafA (Artner et al., 2010). Further, MafB activates genes involved in mature endocrine function, including those involved in glucose sensing, vesicle maturation and insulin secretion (Artner et al., 2010). MafB appears more dominant in early pancreatic development, but as the endocrine pancreas further develops into functional islets, MafA assumes a more dominant role.

There are seven Rfx (regulatory factor X; Rfx1–Rfx7) in mammals (Reith et al., 1994; Emery et al., 1996; Aftab et al., 2008). Rfx transcription factors are active in islet development (Ait-Lounis et al., 2010); Rfx3, like Rfx6, is implicated in the development of pancreatic endocrine cells, including β-cells (Ait-Lounis et al., 2007). β-Cell development was impaired from e15.5 onwards, characterized by the accumulation of β-cell precursors and defective β-cells with reduced insulin, Glut2 and glucokinase expression (Ait-Lounis et al., 2007). Rfx3 is required for the differentiation and function of mature β-cells (Ait-Lounis et al., 2010). Rfx6 is mandatory for the development of pancreatic endocrine cells (Smith et al., 2010). In mice, Rfx6 is expressed in the definitive endoderm early during development and is then restricted to the gut and pancreatic bud, re-activated in endocrine progenitor cells, and ultimately restricted to islets in adults (Smith et al., 2010). Further, Rfx6 is expressed in human pancreatic tissue with an expression pattern consistent with that of Rfx6 in adult mice (Smith et al., 2010). It is evident that Rfx transcription factors play prominent roles in defining the growing pancreas and in maintaining functional islets.

The chromatin modification factor, Hdac1, is widely expressed during embryonic development and is required for multiple processes of endodermal organogenesis (Noel et al., 2008). Hdac1 plays a crucial role in hepatic, pancreatic and foregut organogenesis in the zebrafish embryo (Noel et al., 2008). Further, Hdac1 is required for islet aggregation, either within islet cells or in neighbouring tissues promoting this process (Noel et al., 2008). Alternatively, Hdac1 may repress ectopic endocrine cell formation in endodermal tissue anterior to the main islet (Noel et al., 2008).

Hnf6 may be necessary to maintain sufficient levels of Ngn3 within presumptive endocrine progenitor cells to ensure their endocrine fate (Zhang et al., 2009). Since Hnf6 is rapidly down-regulated in committed endocrine cells, this suggests that some signal, specific to endocrine cell lineage, is involved in silencing Hnf6 (Zhang et al., 2009).

Ngn3 is a bHLH transcription factor critical for the specification of endocrine cells in the islets (Villasenor et al., 2008). Both Ngn3 transcript and protein expression occur in two distinct temporal waves, the first occurring early from approx. e8.5–11.0, and the second initiating at approx. e12.0 (Villasenor et al., 2008). Strikingly, this observed biphasic expression correlates with the first and second transitions, which encompass two distinct waves of embryonic endocrine differentiation (Villasenor et al., 2008).

Blocking Ngn3 expression in the pancreas at e11.5 resulted in reduced endocrine differentiation, no change in the number of amylase-positive acinar cells and an augmentation in the pool of undifferentiated epithelial-ductal cells (Prasadan et al., 2010). Endocrine differentiation can be later rescued in this epithelial-ductal population by restoring Ngn3 expression suggesting that endocrine-committed progenitor cells in the developing pancreas may retain the ability to differentiate into endocrine cells (Prasadan et al., 2010). Pax6 serves as a general marker of all early endocrine cells in the developing pancreas (Prasadan et al., 2010). Ngn3 antisense treatment for 7 days in culture significantly reduced Pax6 expression compared with control oligo-treated pancreas (Prasadan et al., 2010). Pax6 was restored to normal levels after the Ngn3 expression was rescued (Prasadan et al., 2010). The ability to recover endocrine differentiation suggests that the requirement for Ngn3 expression in endocrine differentiation persists later into development (Prasadan et al., 2010).

Other transcription factors play different roles in defining pancreatic fate. Isl1 expression in the dorsal mesenchyme is required for its maintenance and, indirectly, for exocrine pancreas differentiation, whereas Isl1 expressed in pancreatic progenitor cells is necessary for endocrine pancreas differentiation (Bonal and Herrera, 2008). Hex is not required for the specification of the ventral pancreatic fate, but for the proper location of pancreatic progenitor cells in the leading edge of the ventral embryonic endoderm, which can then escape the influence of mesenchymal inhibitors (Bonal and Herrera, 2008). Genetic cell tracing analyses confirmed that Ptf1a is a bona fide pancreatic marker, even better than Pdx1, which is expressed earlier but not exclusively in the pancreas (Bonal and Herrera, 2008). Prox1 is expressed in nearly all pancreatic progenitor cells in early development (Wang et al., 2005). Prox1 is first expressed at e7.5 in endodermal cells and promotes the endocrine fate during the commitment of the different pancreatic lineages (Bonal and Herrera, 2008).

Several novel factors have been found in the developing pancreas. Ndrg4A2 (n-Myc downstream regulated gene-4A2), which is expressed in pancreatic ducts, may also be involved in the regulation of pancreatic cell differentiation (Wang and Hill, 2009). Cited 2 {CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300-interacting transactivator-2} has a glutamic acid/aspartic acid-rich C-terminal domain 2 and encodes a transcriptional modulator and is expressed in the pancreas (Hayata et al., 2009). Ptf1a (Pctk1) has been shown to play a role in membrane trafficking from endoplasmic reticulum to Golgi (Palmer et al., 2005), suggesting that Pctk2 may be involved in a similar secretory pathway during pancreas development (Hayata et al., 2009). Reep5 (receptor accessory protein 5) is a member of the Reep family (Hayata et al., 2009) with Reep1 involved in cell surface expression of odorant receptors that belong to the GPCR (G-protein-coupled receptor) superfamily (Saito et al., 2004). Specific expression of Reep5 was recently demonstrated in the pancreas (Hayata et al., 2009). CTGF (connective tissue growth factor) is expressed in multiple cell types within the pancreas and acts in either an autocrine or paracrine manner during pancreas development (Guney and Gannon, 2009).

At the mid-tail bud stage, Stxbp6 (syntaxin binding protein 6) expression is first detected in the ventral pancreas and later in both pancreatic buds (Hayata et al., 2009). Stxbp6 expression is maintained in the cement gland, cranial ganglia and pancreas (Hayata et al., 2009). While Btg1 (B-cell translocation gene-1) is expressed in the adult pancreas, and weakly in the stomach and the liver (Prevot et al., 2001), the function of Btg1 in the embryonic pancreas remains unknown (Hayata et al., 2009).

Genomic hypomethylation caused by disrupted Dnmt1 (DNA methyltransferase 1) activity is correlated with a greater capability to form de novo β-cells in response to ablation (Anderson et al., 2009). Increased β-cell regeneration in Dnmt1-depleted zebrafish may result from reprogramming of terminally differentiated pancreatic cells, the facilitation of β-cell production from multipotent progenitors or simply an increased capacity for endocrine cell differentiation in the absence of exocrine tissue (Anderson et al., 2009).

6. Endocrine pancreas differentiation

β-Cell terminal differentiation proceeds normally in cells that lose Hnf6 subsequent to activation of the Ngn3 promoter (Zhang et al., 2009). Hnf6 functions specifically during the initial steps of endocrine specification (Zhang et al., 2009).

Sox9 is essential for normal pancreatic development by either promoting or inhibiting the transition from pancreatic progenitor cells to a specific lineage, dependent on the pathway (McDonald et al., 2009). Sox9 is a multipotent progenitor cell marker in the developing pancreas (Prasadan et al., 2010). It is expressed in a subset of Pdx1-positive epithelial cells that are both Notch-responsive and mitotically active, possibly regulating cell proliferation and differentiation of both exocrine and endocrine tissues (Seymour et al., 2007). Sox9 expression, however, is excluded from committed cells (Seymour et al., 2008). Blocking Ngn3 expression in embryonic pancreata in vitro resulted in a significant inhibition of endocrine differentiation; however, there was no effect on the number of amylase-positive cells, suggesting that, after an initial endocrine commitment, these pre-endocrine cells cannot be reprogrammed to become exocrine cells (Prasadan et al., 2010). Lack of an effect of Ngn3 antisense on amylase expression, together with the demonstrated ability to rescue insulin and glucagon expression in pancreata previously treated with Ngn3 antisense, suggest that these pancreatic progenitor cells are already endocrine-committed (Prasadan et al., 2010). Thus blocking Ngn3 in these cells may only prevent Ngn3-dependent endocrine differentiation (Prasadan et al., 2010).

TGFβ signalling controls the number and differentiation of β-cells by promoting their differentiation (Bonal and Herrera, 2008). CTGF may act by modulating the TGFβ signalling pathway, which has also been implicated in endocrine differentiation (Guney and Gannon, 2009). Specifically, endocrine progenitor cells at e11.5 in the pancreas appear to retain their endocrine differentiation potential for at least 3 days when Ngn3 expression is transiently inhibited (Prasadan et al., 2010). The presence of a normal number of glucagon cells in a pancreas in which the Ngn3 expression was blocked for the final 4 days of culture suggests that the cells that initially expressed Ngn3, but were inhibited by the antisense, had committed towards an endocrine fate, but further expression of Ngn3 was required for β-cell differentiation (Prasadan et al., 2010).

At e12.5–14.5, a subpopulation of cells with up-regulation of Pdx1 expression but without co-expressing insulin markers indicate a differentiating islet cell (Wescott et al., 2009). This subpopulation of cells has characteristic features suggestive of branching, such as extension outward into the mesenchyme and away from adjacent epithelial cells (Wescott et al., 2009).

Ngn3-induced islet neogenesis in the liver is the result, not of transdifferentiation, but of transdetermination, i.e. by the switching of progenitor cells from one determined state to another closely related one (Yechoor et al., 2009). Thus not only is Ngn3 necessary, it is also sufficient in inducing islet formation in mice in vivo and progenitor cells may represent an attractive receptive lineage for islet neogenesis (Yechoor et al., 2009).

In the pancreas, Pax4 and Pax6 are initially expressed throughout the early pancreatic bud, but with further differentiation into exocrine and endocrine components, expression becomes restricted to islet cell precursors and a subset of normal adult islet cells (Long et al., 2010). Pax6 is necessary for the expansion and maturation of all endocrine cells and for islet organization (Bonal and Herrera, 2008). As Pax8 has an established role in the organ-ogenesis of other tissues, the discovery of Pax8 in adult islet cells suggests that it may also be a marker for terminal differentiation of endocrine cells in the pancreas or may play a role in endocrine cell development (Long et al., 2010). The Pax transcription factors have a role throughout pancreas development, from organogenesis to differentiation and the establishment of functional islets.

NeuroD1 (neurogenic differentiation 1) is another critical transcription factor that promotes islet cell differentiation (Shimoda et al., 2010) and is suggested to differentiate progenitor cells or even other organs' cells into β-cells (Kojima et al., 2003; Noguchi et al., 2006). NeuroD1, Pdx1 and MafA directly bind to the insulin gene promoter and function as critical transcription factors in pancreatic β-cell differentiation and maintenance (Kaneto et al., 2009). The combination of NeuroD1, Pdx1 and MafA markedly induces insulin biosynthesis in various non-β-cells and therefore is a useful tool to efficiently induce insulin-producing surrogate β-cells (Kaneto et al., 2009).

The early fate choice of pancreatic progenitor cells between the endocrine and acinar cell lineage is restricted by cross-repressive interactions between the transcription factors Nkx6.1/Nkx6.2 (Nkx6) and Ptf1a (Schaffer et al., 2010). Expression of Nkx6.1 favours an endocrine over a ductal cell fate choice (Schaffer et al., 2010). Nkx6.1 may first establish a bipotential ductal/endocrine compartment by excluding Ptf1a and subsequently promote an endocrine fate choice in this secondary progenitor domain (Schaffer et al., 2010).

7. Establishment of the functional endocrine pancreas

Organogenesis, angiogenesis and innervation are required to establish endocrine islets capable of regulating glucose homoeostasis. Angiogenic and neural factors involved in pancreatic organogenesis are displayed in Table 2.


Table 2 Critical factors associated with angiogenesis and innervation in the developing endocrine pancreas

Process   Factor
Angiogenesis   FGF10
  Haemopoietic-derived cells
  MSC
  VEGFA
Innervation   GDNF
  NGF



7.1. Angiogenesis

7.1.1. Overview

Endocrine pancreatic organogenesis also requires islet angiogenesis and innervation to establish a viable micro-organ. The two major processes involved in embryonic and extra-embryonic blood vessel formation are angiogenesis and vasculogenesis (Carmeliet, 2003). Angiogenesis is the budding and branching of vessels from pre-existing vessels and plays a critical role in the morphogenesis of several tissues during embryonic life (Lazarovici et al., 2006), while vasculogenesis refers to the de novo differentiation of endothelial cells from the mesoderm and organization of endothelial progenitors into a primitive vascular plexus (Carmeliet, 2003). Differentiation of the endocardium of the heart and development of larger vascular networks occur by vasculogenesis, while other organs such as the brain and kidney appear to be vascularized primarily by angiogenesis (Brissova et al., 2006). Pancreatic vascularization occurs mainly by angiogenesis. Three major families of angiogenic factor receptor tyrosine kinases, VEGFRs (VEGF receptors), Tie receptors and Eph receptors, play prominent roles in regulating vasculogenesis and angiogenesis in both development and disease (Yancopoulos et al., 2000; Brantley-Sieders and Chen, 2004; Pasquale, 2005). Endothelial cells develop and localize around the pancreatic epithelium during pancreas development (Pierreux et al., 2010). When the epithelial bud undergoes branching remodelling, the endothelial cells become preferentially positioned close to the trunk cells of the epithelium, while remaining at a distance from the branch tips where exocrine cells differentiate (Pierreux et al., 2010).

7.2. Angiogenic factors contributing to islet development and function

7.2.1. VEGFA

VEGF is a mitogen that specifically acts on endothelial cells and has various effects, including mediating increased vascular permeability, inducing angiogenesis, vasculogenesis, endothelial cell growth, promoting cell migration and inhibiting apoptosis (Fiore et al., 2009). The central role of VEGF in angiogenesis is dependent on its ability to co-ordinately regulate, through the activation of signalling pathways downstream from its receptor VEGFR2, multiple endothelial functions that include survival, proliferation, differentiation, adhesion, migration and vascular permeability (Lazarovici et al., 2006). Endocrine cell–endothelial cell communication is reciprocal, since early differentiating endocrine cells produce angiogenic factors including the VEGF family member VEGFA and Ang1 (angiopoietin 1) (Brissova et al., 2006). The development of islet vasculature occurs concomitant with islet morphogenesis, as coalescing endocrine cells are adjacent to blood-perfused capillaries before being assembled into the final structure of mature islets (Brissova et al., 2006).

There is a close association between vascular endothelial cells and the pancreatic epithelium that is initiated early in pancreas morphogenesis and progresses throughout development (Guney and Gannon, 2009). VEGFA plays a unique role in islet vascularization and is expressed by developing endocrine cells as early as e13.5, and is required for the formation of the normal vasculature within the islet (Brissova et al., 2006). Reciprocally, endothelial cells play an inductive role in endocrine development (Lammert et al., 2001). β-Cell-specific inactivation of VEGFA results in a substantial loss in islet vessel density, vessel size and vascular permeability, whereas vasculature in exocrine tissue remained intact (Brissova et al., 2006). Therefore islet vascularization appears sensitive to the level of VEGFA expression (Brissova et al., 2006).

During pancreatic development, VEGFA is secreted by the epithelium and is required for recruitment, development and function of pancreatic endothelial cells (Pierreux et al., 2010). Further, endothelial cells signal back to the growing epithelium in order to repress Ptf1a and exocrine differentiation (Pierreux et al., 2010). Endothelial cells are also required to maintain the centrally located endocrine progenitors (Pierreux et al., 2010). VEGFA signalling is required but not sufficient for Sox9 expression, with VEGFA signalling in endothelial cells required to maintain the Sox-positive progenitor pool and its endocrine derivatives (Pierreux et al., 2010).

7.2.2. FGF10 (fibroblast growth factor 10)

Signals from the dorsal aorta may act in part by influencing epithelial–mesenchymal interactions as the endothelium induces expression of FGF10, a mesodermally derived factor required for proliferation of pancreatic progenitor cells and branching of the epithelium (Jacquemin et al., 2006). The close vicinity of blood vessels and the pancreatic endoderm is essential for pancreatic development (Bonal and Herrera, 2008). At e8.75–9.0, the two dorsal aortae fuse, separating the notochord from the pancreatic endoderm (Bonal and Herrera, 2008). The survival signals from the aorta allow the dorsal mesenchyme to secrete FGF10, which in turn promotes dorsal pancreas development (Jacquemin et al., 2006).

7.2.3. Haemopoietic-derived cells

Haemopoietic-derived cells also play a role in establishing pancreatic vascularization. Pancreatic vascular endothelium can induce β-cell differentiation, while also providing key signals via a shared basement membrane for β-cell growth and function (Lammert et al., 2003a; Nikolova et al., 2006). Paracrine interplay within the islet allows synthesis of VEGF from the β-cells to contribute to endothelial cell proliferation, whereas a reciprocal production of HGF by the endothelial cells promotes β-cell growth (Johansson et al., 2006). In rats, haemopoietic-derived cells were present within the pancreas at all ages between 10 and 130 days, being most abundant in the neonate (Chamson-Reig et al., 2010). The haemopoietic-derived cells included the expected lineage progeny such as endothelial cells and monocytes/macrophages (Chamson-Reig et al., 2010). However, the localization and morphology of haemopoietic lineage cells within the pancreas were not random and were compatible with a functional interaction with endocrine cells or their progenitors (Chamson-Reig et al., 2010). Genetic tagging has identified haemopoietic lineage cells during normal postnatal pancreatic development represented by four main progeny phenotypes: endothelial cells, macrophages, epithelial cells and Pdx1-expressing cells that demonstrate differential distributions within islets and around ducts (Chamson-Reig et al., 2010).

7.2.4. MSC (mesenchymal cells)

MSC enhance pancreatic vascularization. Islet quality improvement was achieved by an MSC-derived trophic factor as islets cultured in MSC-conditioned media facilitated the regulation of blood glucose at normoglycaemic concentrations, with the recipient mice demonstrating superior weight gain and glucose tolerance capacities (Park et al., 2010). Histological analysis revealed an increase in the intensity of insulin staining and significantly enhanced blood vessel formation in islets cultured with MSC-conditioned media (Park et al., 2010). Well-developed blood vessel structures can improve insulin secretion through the interaction between the in vitro evidence demonstrating differences in VEGF and angiogenesis-related signalling and β-cell function enhancement (Johansson et al., 2006). The increased blood vessel formation of MSC-conditioned media-cultured islet grafts is likely caused by a trophic factor related to MSCs, which induces signals involved in β-cell survival and intra-islet endothelial revascularization and endothelial cells (Park et al., 2010).

7.2.5. Other factors

NCAM (neural cell adhesion molecule) is a member of the Ig superfamily that mediates Ca2+-independent cell–cell and cell–substratum interactions by homophilic and heterophilic interactions; further NCAM is required for cell type segregation during islet organogenesis (Olofsson et al., 2009). Fibrin plays a role in early neovascularization and support to sustain new blood vessel development by forming a matrix to maintain an intact three-dimensional structure of human islets, thereby reducing environmental stress on the islets (Sabek et al., 2010). GJA7 (gap junction membrane channel protein α-7) and PLXND1 (plexin D1) may play roles in vascular patterning during pancreas development (Hayata et al., 2009).

7.3. The role of vascularization in mature islet function

The vascular network within islets consists of vessels that are wider, more numerous and more tortuous compared with the surrounding exocrine tissue (Brissova et al., 2006). The density of the capillary network in the islets is approx. 10 times higher than that of the surrounding tissue (Henderson and Moss, 1985; Kuroda et al., 1995), indicating the requirement of higher oxygen levels for normal islet cell function and survival (Ito et al., 2010). Postnatally islets develop an abundant glomerular-like microcirculation system that ensures that no portion of an islet is more than one cell away from arterial blood (Bonner-Weir, 1988). This arrangement optimizes the condition for their supply of oxygen and nutrients to the islet cells, their metabolic sensing and the distribution of secreted hormones to target organs in order to maintain glucose homoeostasis (Lau et al., 2009). The islet capillary network undergoes considerable remodelling in the early postnatal period, which coincides with a rapid increase in islet mass after birth (Georgia and Bhushan, 2004). Adult islets are highly vascularized and proper vascular organization and function are required for maintaining glucose homoeostasis in mice (Guney and Gannon, 2009).

Several angiogenic and growth factors contribute to establishing functional islets. Further studies are required to determine how these factors interact with transcription and other factors established in the hierarchy of islet development, differentiation and maturation.

7.4. Innervation

7.4.1. Overview

The islets represent a complex regulatory system involving neurocrine, paracrine and endocrine signalling coupled with modulation of blood flow to regulate the effects of humoral signals (Barreto et al., 2010). The complexity and redundancy observed likely reflect an intricate regulation of important physiological functions at a local and systemic level (Barreto et al., 2010). The islets are densely innervated (Ramnath and Bhatia, 2006) involving the central and autonomic nervous systems with afferent and efferent signalling with the vagus nerve as the major regulatory pathway (Cabrera-Vasquez et al., 2009; Barreto et al., 2010). The pancreas is richly innervated by preganglionic vagal neurons (Ahren et al., 1986; Berthoud and Powley, 1990; Kirchgessner and Gershon, 1990; Brunicardi et al., 1995). Autonomic nerves synapse on to intrapancreatic ganglia clusters of neurons that are spread in a connective plexus throughout the pancreas in mice, rats, cats, rabbits and guinea pigs (Coupland, 1958; King et al., 1989; Kirchgessner and Pintar, 1991; Ushiki and Watanabe, 1997). Sympathetic neural cell bodies are located in the superior mesenteric and celiac ganglia and are components of the splanchnic nerve and parasympathetic innervation is derived from the vagus nerve (Salvioli et al., 2002).

Enteropancreatic neurons between the pancreas and the gastrointestinal tract are reported to mediate enteropancreatic reflexes that are important for the intestinal phase of pancreatic exocrine secretion (Singer et al., 1980; Singer, 1983; Kirchgessner and Gershon, 1990). The neurotransmitters acetylcholine and norepinephrine (noradrenaline) are the major pancreas transmitters, with several peptides as co-transmitters that include VIP (vasoactive intestinal peptide), PACAP (pituitary adenylate cyclise activating polypeptide), substance P and galanin (Barreto et al., 2010). CCK (cholecystokinin) has also been localized in islet nerves (Rehfeld et al., 1980).

7.4.2. Islet innervation

Insulin secretion from islets promotes glucose absorption by the liver, affecting all islets (Fendler et al., 2009) and has a synchronizing effect on the islet population (Pedersen et al., 2005). A potential synchronizing mechanism is neural input from intrapancreatic ganglia (Ahren, 2000). The ganglia have been shown to be electrically excitable when autonomic nerve trunks are stimulated in cats (King et al., 1989). Further, in vivo and in vitro vagal stimulation promotes glucose-dependent insulin release from the pancreas (Bloom and Edwards, 1980; Ahren and Taborsky, 1986; Nishi et al., 1987; Berthoud and Powley, 1990). Ganglia are often found in the proximity of islets and provide innervation (Coupland, 1958; Morgan and Lobl, 1968; Persson-Sjogren et al., 2001). It is not necessary for the whole islet population of a pancreas to be innervated to produce a significant oscillatory insulin response (Fendler et al., 2009) as only ∼35% innervation was required to produce a regular oscillatory insulin signal with a period similar to that measured in vivo in mice (Nunemaker et al., 2005) and humans (Mao et al., 1999; Song et al., 2000).

Islet-innervating nerves include peptidergic, cholinergic, adrenergic and GABAergic fibres (Ahren, 2000; Kiba, 2004). Some of these nerves directly synapse with endocrine cells, influencing their activity (Tsui et al., 2008). Sympathetic innervation plays important roles in the physiology and pathophysiology of the endocrine pancreas; norepinephrine and epinephrine inhibit insulin secretion, but stimulate glucagon secretion (Ahren, 2000). Moreover, sympathetic innervation of islets is altered in animal models with insulin resistance and Type 2 diabetes (Ahren, 2006). Using NPY (neuropeptide Y) as a marker, a loss of sympathetic nerves was demonstrated within the islets of NOD (non-obese diabetic) mice that had been diabetic for only 3 weeks (Taborsky et al., 2009). Because activation of islet sympathetic nerves stimulates glucagon secretion (Marliss et al., 1973), this islet nerve loss has the potential to remove a significant α-cell stimulator that is >both activated during hypoglycaemia (Havel et al., 1988) and contributes to the glucagon response to insulin-induced hypoinsulinaemia in non-diabetic animals (Havel et al., 1996).

7.5. Neural factors contributing to islet development

7.5.1. GDNF (glial-derived neurotrophic factor)

GDNF is a factor produced by glial cells and is essential for the development of the enteric nervous system (Mwangi et al., 2010). GDNF enhances Pdx1, Ngn3, NeuroD1/2 and Pax4 gene expression in embryonic mouse pancreata and β-cell proliferation in both embryonic and postnatal mouse pancreata (Mwangi et al., 2010). Further, GDNF can enhance the binding of the transcription factors NeuroD1/2 and Sox9 to the Pdx1 promoter to stimulate the gene and influences β-cell development (Mwangi et al., 2010).

7.5.2. NGF (nerve growth factor)

NGF was identified as a potent neurotrophic factor for sympathetic and sensory neurons (Levi-Montalcini, 1952) and also has direct involvement in angiogenic processes with a role in neovascularization, along with VEGF and FGF (Lazarovici et al., 2006). The biological actions of NGF are mediated through two classes of cell-surface receptors, namely the p75 NTR (p75 neurotrophin receptor), common to all members of the neurotrophin family, and the TrkA (tropomyosine kinase-related receptor A), belonging to the tyrosine kinase-neutrotophin receptor family (Kaplan and Miller, 1997). These biological actions are exerted on a cellular level influencing macrophages, mast cells (Aloe et al., 1999), fibroblasts (Pozza et al., 2000), osteoblasts (Mogi et al., 2000), cells of the haemopoietic immune system (Bracci-Laudiero et al., 2003) and endothelial cells (Hammes et al., 1995), inducing proliferation of umbilical cord, brain capillary, choroidal and dermal microvasculature endothelial cells (Lazarovici et al., 2006). NGF has powerful synaptotrophic effects on excitory and inhibitory synaptic inputs to motoneurons that control firing patterns and discharge characteristics (Davis-Lopez de Carrizosa et al., 2010).

NGF is synthesized by the peripheral targets of NGF-dependent sympathetic neurons and NGF concentrations are proportional to their innervation density (Bjerre et al., 1975). Adult β-cells produce and secrete NGF (Rosenbaum et al., 1998) and express both p75 NTR and TrkA receptors during their lifespan; NGF is important for normal islet morphogenesis during prenatal life (Kanaka-Gantenbein et al., 1995a, 1995b) and has trophic effects on β-cell survival, maturation and insulin secretion (Rosenbaum et al., 2001; Navarro-Tableros et al., 2004, 2007; Hiriart and Aguilar-Bryan, 2008) that are controlled by autocrine or paracrine mechanisms (Rosenbaum et al., 1998). NGF may be trophic for sympathetic innervation and survival in islets, as observed in other organs (Lara et al., 1990; Carlson and Craig, 1995; Dissen et al., 2002), and its secretion in adult cells could also be important for sympathetic fibre maintenance throughout life (Cabrera-Vasquez et al., 2009). Sympathetic control of insulin release has been relatively well characterized, with adrenalin inhibiting insulin and stimulating glucagon secretion (Dunning and Taborsky, 1991). Sympathetic islet innervation also contributes to the glucagon response after insulin-induced hypoglycaemia (Benthem et al., 2001; Persson-Sjogren, 2001).

7.6. The role of the sympathetic nervous system in islet development and function

In early development, islets receive extensive innervation by sympathetic fibres that are refined later in development (Cabrera-Vasquez et al., 2009). A progressive increase in the relative area occupied by TH (tyrosine hydroxylase)-positive innervation, to identify sympathetic nerve fibres, in the islets was observed between e19 to postnatal day 20 (Cabrera-Vasquez et al., 2009). Interestingly, this parameter decreased between postnatal day 20 and adulthood in the periphery of the islets, but increased in the central area (Cabrera-Vasquez et al., 2009). When islets are transplanted to the kidney capsule or intraportally, they develop sympathetic innervation (Gardemann et al., 1994; Myrsen et al., 1996). Moreover, when purified β-cells are transplanted to the kidney capsule, grafts are progressively innervated mainly by TH-containing nerve fibres; these observations suggest that factors produced by β-cells may mediate islet neurotrophism (Myrsen et al., 1996).

Sympathetic innervation is subjected to strong ontogenetic remodelling during pancreatic development (Cabrera-Vasquez et al., 2009). There is a trend of innervation to increase from the perinatal period to postnatal day 20 (Cabrera-Vasquez et al., 2009). After this time, the amount of innervation within the islet decreases (Cabrera-Vasquez et al., 2009). Early hyper-innervation followed by a late elimination of redundant axons mediated through competitive interactions is commonly seen in peripheral targets (Silva et al., 2002; Deppmann et al., 2008). These morphological and functional data suggest that NGF plays a pivotal role in islet morphogenesis, remodelling and maintenance of sympathetic innervation and development of the vasculature and maturation of α- and β-cells (Cabrera-Vasquez et al., 2009).

8. Perspectives

Despite only representing ∼1–2% of the total pancreatic mass, islets are one of the most studied micro-organs due to the global significance of diabetes. Pancreatic ontogeny shares ancestry with the digestive system, while the islets are linked to a neuroendocrine lineage. Pancreatic organogenesis is strongly associated with angiogenesis and innervation throughout development. Tightly regulated and co-ordinated angiogenesis and innervation during pancreatic organogenesis is critical for normal islet development and function. Studies that focus on these three core aspects of establishing a functional endocrine unit will increase our knowledge of the mechanisms involved in these processes and identify areas for therapeutic intervention.

Exocrine (acinar) and ductal cell differentiation follow their own differentiation patterns during development and maturation. Specifically the endocrine cells differentiate after activation of a cascade of transcription factor networks to form specialized hormone-secreting cells arranged as islets. The transcription factor networks specific for embryogenesis, organogenesis and endocrine islet cell differentiation vary in constituency; however, some overlap of factors involved in early development recur as the pancreas matures. Transcription factors play a major role in destining the fate of the pancreas during embryogenesis, organogenesis and in defining (differentiating) the endocrine pancreas. The endocrine pancreas is highly vascularized and innervated. Further, adipogenic, neural, growth, co-factors and other factors all contribute to establishing functional islets. Collectively these factors act in a co-ordinated manner throughout pancreatic development to produce functional islets. The overlapping nature and hierarchy of these factors during development remains to be fully elucidated.

Angiogenesis and neurogenesis are distinct, yet related, biological processes that primarily occur during embryonic development but continue to play an important physiological role throughout life (Lazarovici et al., 2006). For example, sensory nerves determine the pattern of arterial differentiation and blood vessel branching (Miller, 2002). Further, VEGF stimulates neurogenesis (proliferation), increases survival and promotes growth (neurotrophic factor) for a variety of neuronal and glial cell types of different species of nervous tissue origin (Lazarovici et al., 2006). VEGF production by sympathetic neurons is strongly stimulated by NGF administration, suggesting that NGF promotes neuron-induced angiogenesis by stimulating VEGF production (Calza et al., 2001). This illustrates the close angio- and neuro-genic association particularly during developmental phases.

Pancreatic development and its angiogenesis are closely associated (Fetita et al., 2006) with blood vessel endothelium essential for cell differentiation and pancreatic morphogenesis through signalling molecules, while endothelial cells induce islet formation (Lammert et al., 2001, 2003a). Further, newly formed islets express high levels of VEGFs that, through paracrine signalling, regulate proliferation and differentiation of vascular endothelium to vascularize islets (Lammert et al., 2003b). Importantly during embryonic and fetal development there is cross-talk and interdependence between the vascular and neuronal systems (Weinstein, 2005). This cross-talk likely extends to the pancreas, as elements of both systems, namely angiogenic and neural factors like VEGF and NGF respectively promote development of the pancreas and are influenced or influence transcription factors involved in the pancreatic developmental process. In addition, GDNF enhances Pdx1, Ngn3, NeuroD1/2 and Pax4 gene expression in embryonic mouse pancreata and β-cell proliferation in embryonic and postnatal mouse pancreata (Mwangi et al., 2010). Further, GDNF was shown to enhance the binding of the transcription factors NeuroD1/2 and Sox9 to the Pdx1 promoter to stimulate the gene (Mwangi et al., 2010). These recent findings provide evidence of a mechanism by which GDNF can influence β-cell development (Mwangi et al., 2010).

The interrelationships of pancreatic development, angiogenesis and innervation need to be established to advance our understanding of pancreatic development. Figure 1 displays the association of pancreatic development, angiogenesis and innervation. Pancreatic development progresses from organogenesis of the pancreas, to differentiation of cells, including the islet cells, ultimately establishing a functional endocrine micro-organ, the islets characterized by insulin-secreting β-cells and the other hormone-secreting cell types. Throughout these stages of generating functional β-cells, key factors are implicated for normal pancreatic development. The processes of angiogenesis and innervation occur as functional islets develop, all regulated by critical factors. Upon injury, transplantation and metabolic states such as insulin resistance, metabolic syndrome and Type 1 and 2 diabetes, pancreatic transcription factors, angiogenic, neural, growth and other essential factors are mobilized and potentially synergized to restore islet homoeostasis reflected by a functional islet phenotype. The processes of pancreatic development, angiogenesis and innervation are not linear, but influenced by the dynamics of the pancreas such as metabolic demands and likely act in continuum throughout life. In the mature pancreas, this triadic association still remains, but with reduced capacity relative to early development. Injury to the pancreas and diabetes serve as stimuli to set off the angiogenic and neural processes to support and progress along with pancreatic development. Upon β-cell demise, differentiation of progenitor cells is triggered in an attempt to restore the β-cell population and phenotype. Differentiation of progenitor cells residing in the pancreas and other tissues into functional β-cells are a source for restoring functional β-cells after insult.

Studies have largely focused on the lineage of the endocrine pancreas. However, since the endocrine pancreas shares some ancestry of neuroendocrine origin, angiogenic factors are implicated in endocrine pancreatic development and other factors involved in the development of other organs may also be involved, the myriad of these vast number of factors, many yet unidentified, remains a major task for elucidation. Both the adult liver and pancreas are suggested to contain cells with epigenetic memory of their common embryonic origin (Zaret and Grompe, 2008). The existence of potential β-cell precursors in the adult liver is of clinical interest (Zaret and Grompe, 2008). Since pancreatic exocrine cells greatly outnumber β-cells, the reprogramming of exocrine cells to generate functional β-cells in vivo by viral delivery of the developmental transcription factors Pdx1, Ngn3 and MafA remains a prospect for investigation (Zhou et al., 2008). Reflecting on the challenges of treating Type 2 diabetes, the potential exists that further definition of the footprint of collective factors involved in endocrine pancreas specification, from embryogenesis through to maturation, will yield vital clues in our combating of this major global epidemic. ADFP (adipose differentiation-related protein), which is localized on the surface of lipid droplets, is expressed in β-cells, influenced by nutritional cues and plays a role in fatty acid metabolism in β-cells (Faleck et al., 2010). Preliminary analysis in culture media of peripancreatic adipose tissue after 6 months of treatment with 20% sucrose solution in drinking water revealed an increase in the release of IL (interleukin)-10, IL-6, TNFα (tumour necrosis factor α) and NGF (Duhne et al., 2009). Low protein (Snoeck et al., 1990; Dahri et al., 1995; Dumortier et al., 2007), high carbohydrate (Petrik et al., 2001) and high fat (Cerf et al., 2005, 2007) dietary intervention during early life all induce adverse effects on islet development. These studies implicate adipose-derived factors and diet in influencing islet organogenesis. The overlap of angiogenic factors, neural factors, pancreatic transcription factors and other factors involved in regulating pancreatic development will advance our understanding of Type 2 diabetes and aid our discovery of novel therapeutic agents designed to maintain functional β-cell populations.

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Received 1 November 2010/27 January 2011; accepted 3 March 2011

Published online 27 September 2011, doi:10.1042/CBI20100780


© The Author(s) Journal compilation © 2011 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)