Development of the Endocrine and Exocrine Pancreas

Acknowledgment

Some content was based on a previous chapter by Steven L. Werlin, MD, and Alan N. Mayer, MD.

Embryology and Histogenesis of the Human Pancreas

The pancreas is a multifunctional organ consisting of three major tissues: exocrine tissue, endocrine islets, and epithelial ducts ( Fig. 85.1 ). The exocrine compartment contains the largest proportion of cells within the adult pancreas and is composed of acinar cells, which synthesize and secrete digestive enzymes. The endocrine pancreas is composed of hormone-producing cells organized within the islets of Langerhans that are responsible for maintaining glucose homeostasis. These cells constitute approximately 1% to 2% of the adult pancreas. The epithelial ductal cells are also a small, but essential component of the pancreas, and act as a conduit for transporting the acinar enzymes into the intestinal lumen. Although each pancreatic tissue type performs diverse functions, they arise from a common progenitor pool in the foregut endoderm, and the respective developmental programs are closely associated. In this chapter, we will discuss what is known about the coordinated regulation of pancreas development and some of the diseases that are associated with defects in these developmental programs.

Fig. 85.1, The pancreas is a multifunctional, glandular organ nestled between the small intestine and spleen. It is comprised of the exocrine acinar cells that secrete digestive enzymes into the ductal system and endocrine cells that are located within the islets of Langerhans that secrete pancreatic hormones into the blood system.

The pancreas is a unique organ in that it initially develops as two distinct anlagen. In most vertebrates, the dorsal and ventral pancreatic anlagen develop as evaginations of the primitive foregut endoderm ( Fig. 85.2 ). In humans, the larger dorsal anlage, which develops into the tail, body, and part of the head of the pancreas, grows directly from the duodenum and becomes visible by 26 days after conception. The ventral anlage develops by 33 days after conception as two evaginations off the bile duct and eventually contributes to the head of the pancreas. The ventral anlagen are initially paired, with the left lobe ultimately disappearing over time. At approximately 7 weeks gestation, the dorsal and ventral anlagen fuse as the buds develop and the gut rotates. The ventral duct forms the proximal portion of the major pancreatic duct of Wirsung. The dorsal duct forms the distal portion of the duct of Wirsung and the accessory duct of Santorini. Variations in fusion account for the variety of developmental abnormalities of the pancreas, such as anomalous pancreaticobiliary junction, annular pancreas, and pancreas divisum. Pancreas divisum occurs in approximately 10% of the population, and although the majority of individuals are asymptomatic, some suffer from acute or chronic pancreatitis.

Fig. 85.2, Development of the human pancreas. (A) Gestational age 6 weeks. (B) Gestational age 7 to 8 weeks. The ventral pancreas has rotated but has not yet fused with the dorsal pancreas. (C) The ventral and dorsal pancreatic ductal systems have fused.

Histologic examination of the emerging human pancreatic buds reveals predominantly undifferentiated epithelial cells that undergo active growth and branching morphogenesis to form a lobular-tubular pattern surrounded by loose mesenchymal stroma by 9 to 12 weeks after conception ( Fig. 85.3 ). ^, The extracellular matrix present in the stromal compartment provides important signaling molecules that provide instructions for pancreatic differentiation into the different tissue cell types. During human development there is a significant amount of extracellular matrix fibers throughout the pancreas at 9 weeks after conception, and the extracellular matrix has intercalated between large numbers of epithelial cells by 14 to 20 weeks after conception. Collagen I, collagen IV, fibronectin, and laminin are all expressed throughout the pancreas, especially within and around the endocrine cell clusters. In mice, laminin 1 has been shown to play a role in pancreatic duct formation, branching morphogenesis, induction of acinar cells, and enhancement of β cell differentiation.

Fig. 85.3, Pancreatic morphogenesis in mouse and humans. During the stage referred to as the primary transition , the pancreas forms as an epithelial bud surrounded by mesenchymal tissue and extracellular matrix (ECM) . The epithelial bud continues to expand and forms a population of multipotent pancreatic progenitors that can give rise to all cell lineages within the pancreas. At the end of the primary transition, the pancreas undergoes a series of morphologic changes to form a tube-like structure that is divided into distinct domains. The “tip” domain gives rise predominantly to the acinar lineage, whereas the “trunk” domain predominantly gives rise to the ductal and endocrine lineages. During the “secondary transition” stage, pancreatic morphogenesis continues and cell lineage differentiation occurs to form the exocrine, ductal, and endocrine lineages. The endocrine cells begin to cluster into islets.

Several studies have indicated that signals from the vasculature are also required for morphogenesis, growth, and cytodifferentiation of the pancreas. In humans, angiogenesis can be detected between 9 and 22 weeks after conception, at which time maturation is complete and the vasculature is able to provide a functional response. Scanning electron microscopy analysis of the pancreatic vascular architecture between 18 and 25 gestational weeks demonstrated that the lobular structure of the pancreas influences the organization of the microvasculature. Similar to the adult organ, the vascular system of fetal human pancreas has many portal connections, including islet-lobule and islet-duct portal circulations. In mice there is evidence that the vascular endothelial cells modulate both early and late pancreatic development and function to inhibit acinar cell differentiation. ^, Although there is currently no evidence that innervation influences developmental processes, significant innervation of the human pancreas can be detected shortly after vascularization at 13 weeks after conception; within the head of the pancreas there are two primary peaks of nerve growth at 14 and 22 weeks after conception, whereas in the body and tail of the pancreas a single growth peak can be observed at 20 weeks after conception. That study also identified nerve fibers innervating distinct components of the vasculature. Furthermore, a study in mice demonstrated there is coordinated vascularization and innervation of the developing pancreas.

Pancreatic Cell Lineages

Pancreatic endocrine cells that make up the islets of Langerhans are the first lineage to form during vertebrate development. There are four hormone-producing endocrine lineages known to populate the adult islet: α cells (glucagon secreting), β cells (insulin secreting), δ cells (somatostatin secreting), and PP cells (pancreatic polypeptide secreting) (see Fig. 85.1 ). Two additional transient endocrine populations are present in prenatal mouse and human islets: ghrelin-producing epsilon cells and gastrin-producing cells. ^, With the increasing availability of human embryonic and fetal tissue, considerable insight has been gained regarding human endocrine cell development. In humans, rare insulin-positive cells are first apparent scattered throughout the epithelium at 52 days after conception, with isolated glucagon- and somatostatin-positive cells appearing approximately 1 week later at 8.5 weeks gestation. The number of hormone-producing cells significantly increases by 10 weeks to constitute almost 1.5% of the total pancreatic cell population. Between 9 and 10 weeks after conception, PP and epsilon cells are also present. At this stage, insulin-positive cells are the most abundant cell type and begin to aggregate in progressively larger cell clusters. Primitive islet clusters containing a mixture of endocrine hormone-expressing cells are first identifiable at 12 to 13 weeks after conception. Endocrine cells expressing almost all combinations of hormones, in addition to polyhormonal granules, are also frequently observed in the human fetal pancreas. ^, Between 11 and 13 weeks after conception, the highest ratio of insulin and glucagon co-expressing endocrine cell populations is present; however, this number significantly declines by 15 weeks after conception. Ghrelin is the only hormone that does not colocalize with insulin or glucagon. By 21 weeks after conception, single hormone expression has resolved into specific cell types, and the relative ratio of the distinct endocrine cell populations is comparable to that of the adult pancreas. The histologic appearance of the pancreas in an infant born at term is similar to that in the adult.

Exocrine acinar cell differentiation is initiated between 8 and 9 weeks, although at this early-stage exocrine cells are rare and their secretory granules, referred to as zymogen granules, are not yet present. ^, ^, ^, Between 14 and 20 weeks, acinar cells rapidly mature and increase in number; and recognizable zymogen granules become visible by 14 to 16 weeks. ^, As the pancreas matures, the luminal volume decreases and acinar cell volume increases. The amount of connective tissue continues to decrease throughout gestation and in the postnatal period. By 20 weeks gestation, acinar cells contain mature-appearing proteolytic active zymogen granules, well-developed endoplasmic reticulum, and highly developed basolateral membranes. As development proceeds, the amount of stroma continues to decrease, and acinar cells gain a mature appearance. After birth the volume of the exocrine pancreas continues to grow and nearly triples in mass during the first year of life from 5.5 to 14.5 g. The adult pancreas weighs approximately 85 g. During the first 4 months the ratio of acinar cells to connective tissue increases four-fold.

In rodents and humans, the acinar cell population is predominantly mononuclear at birth and acinus formation is due to proliferation of these mononuclear acinar cells and duct cells. Postnatally, acinar cells become progressively binucleate. In the adult, the shape of the mature acinar cell is pyramidal with a basal nucleus. The most prominent organelles in the fasted state are large numbers of zymogen granules, located apically. Abundant rough endoplasmic reticulum and Golgi apparatus are present. Junctional complexes join adjacent acinar cells, and the apical membrane contains abundant microvilli projecting into the lumen. The final three-dimensional structure of the exocrine pancreas consists of a complex series of branching ducts surrounded by grapelike clusters of acinar cells. The ontogeny of cell surface glycoproteins is critical for normal cell-cell interactions and exocrine morphogenesis. ^, Stage-specific polylactosamine (carbohydrate) antigens are dynamically expressed during pancreas morphogenesis as they become preferentially localized to the differentiating cell lineages. These antigens are thought to be important for cell-to-cell communication and recognition.

Duct and centroacinar cells are also found between 14 and 20 weeks after conception. The ductal system constitutes less than 5% of the volume of the exocrine pancreas and only 0.5% of total pancreatic volume. The adult pancreatic ductal system is critical for secreting bicarbonate to dilute and neutralize the enzymes secreted by the acinar cells, in addition to acting as a conduit for these proteins into the intestinal lumen. Glycogen is highly expressed in human ductal cells at 8 weeks after conception, but by 20 weeks the glycogen expression domain shifts primarily into the acinar cell clusters. In the adult pancreas there remains little detectable glycogen expression in ductal or islet cells; however, high glycogen expression can be detected in pancreatic microcystic adenomas. The centroacinar cells lie at the nexus between the acinar cells and the terminal duct epithelium. The origin of the centroacinar cells has not been established; however, in humans, cytokeratin 19–labeled centroacinar cells can be located within amylase-expressing cell clusters between 14 and 20 weeks after conception. Emerging studies in mouse and zebrafish models suggest that centroacinar cells may represent a population of multipotent progenitor cells in the adult pancreas.

Lessons From the Nonhuman Pancreas

Many of the concepts associated with human pancreatic development have historically been based on studies performed in model organisms. Although the relative time spans are different, comparative studies have demonstrated that the histologic, ultrastructural, and molecular developmental stages of rodent and human pancreas are very similar, although some important distinctions have also been identified. ^, ^, Studies in mice have defined three distinct prenatal developmental periods. ^, Initially, a wave of pancreatic progenitor cell proliferation between embryonic day 9.5 and embryonic day 12.5 contributes to the development of a stratified epithelium that is referred to as the primary transition (see Fig. 85.3 ). This is followed by a major wave of cell type differentiation between embryonic day 13.5 and embryonic day 16.5, which is referred to as the secondary transition and describes the differentiation of the major pancreatic lineages. At this time acinar cells form from the extending tip epithelium and large numbers of acini differentiate and continue to proliferate past embryonic day 16.5. The third wave of development, or tertiary transition, occurs postnatally and refers to the adaptive regulation of hormone and enzyme secretion in response to dietary influences. In humans, similar waves of differentiation have been described, although the first phase of differentiation is less well defined. ^, ^.

Conserved Molecular Regulation of Early Pancreas Morphogenesis

Since the mid-1990s there has been progressive elucidation of the intrinsic and extrinsic molecular events that regulate mouse pancreas development ( Fig. 85.4 ; reviewed by Jorgensen and colleagues ). More recently, access to human embryonic and fetal pancreas tissue, human-specific reagents and improved imaging technologies has resulted in demonstration of extensive conservation of many of the molecular pathways in normal human development, especially with regards to the intrinsic transcriptional regulatory programs ( Fig. 85.5 ). It has been more difficult to gain insight into the signaling pathways given the challenges associated with culturing human pancreas.

Fig. 85.4, Origin of the exocrine and endocrine cell lineages in the developing pancreas. This model is based on descriptions from various reports (e.g., see Hebrok and colleagues 42 and Jorgensen and colleagues 35 ). Arrows indicate the sequential activities based on the appearance of specific cell markers in the developing mouse pancreas.

Fig. 85.5, Lineage specification of the human pancreas. The pancreas develops from a common progenitor population and progresses through sequential lineage decisions, similar to those observed in mice, but with some minor differences. The stages and known gene expression data are noted for each major lineage.

As described above, the parenchyma of both endocrine and exocrine glands originates from the endodermal epithelium of the primitive gut tube (see Fig. 85.4 ). Genetic lineage tracing studies in mice have determined that all pancreatic cell types are derived from a common endodermal progenitor cell population. Prior to the evagination of the pancreatic primordia from the primitive gut tube, the molecular programs specifying pancreatic fate have been initiated by the inductive influences of neighboring tissues. During gastrulation the endoderm is subdivided into anterior and posterior domains by signals from the adjacent mesectoderm. Retinoic acid, bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs) play important roles during late gastrulation in localizing the prepancreatic domain, which will respond to subsequent instructive signals that induce pancreatic tissue and growth.

The ventral and dorsal aspects of the endoderm that will give rise to the pancreas are exposed to different local contacts and therefore rely on distinct signaling sources for their induction. The dorsal pancreas is induced by transient contact with the notochord, followed by interposition of the dorsal aorta. Contact with the notochord induces a localized exclusion of an important developmental signaling morphogen, sonic hedgehog (Shh), from the prepancreatic endoderm. This signaling event is necessary for dorsal pancreatic development; ectopic expression of Shh interferes with subsequent patterning events. In humans there appears to be a similar exclusion of Shh from the dorsal epithelium. In mice it has been shown that the dorsal aorta also provides essential signals to promote pancreatic fate, among them vascular endothelial growth factor (VEGF). The ventral pancreas emerges from a noncontiguous endodermal region that is in close association with the hepatic and bile duct endoderm, and is induced by sequential FGF and BMP signals derived from the adjacent cardiac and septum transversum mesoderm.

These early events render the prepancreatic endoderm competent to form pancreatic tissue. The next step involves expression of transcription factors in the endoderm and in the surrounding mesenchyme to further specify a pancreatic fate. The pancreatic and duodenal homeobox 1 gene (Pdx1) is expressed in the prepancreatic endoderm at embryonic day 8.5 in the mouse, and its ablation results in the arrest of pancreatic development shortly after formation of the pancreatic anlagen. In humans PDX1 is expressed comparatively later in development, after the dorsal aortae have fused ; however, similarly to Pdx1 -null mutations in mice, a mutation in the human PDX1 gene was found to cause congenital pancreatic agenesis in an infant.

Basic helix-loop-helix pancreas-specific transcription factor 1 α subunit (Ptf1a) is also expressed throughout the early pancreatic epithelium around embryonic day 9.5, shortly after Pdx1 and in a domain more narrowly approximating the developing pancreas. By embryonic day 12.5, Ptf1a expression becomes restricted to the epithelial tip domain and ultimately the acinar cells, where the gene product functions as part of a trimeric protein complex to regulate exocrine gene expression. Genetic ablation of Ptf1a in mice also results in pancreas agenesis and has provided important insight into the early events of pancreatic specification. By genetic tagging of the mutant cells that would otherwise express Ptf1a , cells lacking Ptf1a were incorporated into the developing duodenum as full-fledged intestinal epithelial cells. This illustrates the developmental plasticity of early pancreatic development and highlights the principle that multiple successive specification events are needed to guide cells from progenitor cells to a fully differentiated state. The expression domain of PTF1A in the developing human pancreas has not yet been determined; however, a recent study using RNA-based fluorescent probes has identified a pool of human multipotential progenitor cells located at the tips of the branching epithelium that co-express SOX9 and PTF1a , similar to rodents. Consistent with its expression within the earliest progenitor population, deletions of PTF1a and mutations in a distal enhancer of PTF1A in humans also cause pancreas agenesis. ^,

The genes encoding two members of the GATA family of transcription factors, GATA4 and GATA6, are also co-expressed with Pdx1 and Ptf1a in early pancreatic endoderm of the mouse; Gata4 subsequently becomes restricted to the exocrine compartment and Gata6 becomes restricted to the ducts and the endocrine compartment. Similarly in humans, GATA4 is expressed in the pancreatic buds and subsequently restricted to the acinar-fated tip cells (see Fig. 85.5 ); GATA6 expression has not been analyzed. Heterozygous mutations in either GATA4 or GATA6 cause pancreas agenesis in humans, with disruption of GATA6 function causing most of the known pancreatic agenesis cases. On the other hand, both GATA4 and GATA6 must be deleted from the pancreatic endoderm domain in mice to cause pancreas agenesis, ^, suggesting there has been a specialization of GATA functions during evolution.

Some of the earliest studies of exocrine pancreas development in rodents highlighted the influence of mesenchymal-epithelial interactions. ^, After initial budding of the pancreatic primordium in mice, FGF10 activates notch signaling to maintain the pool of pancreatic progenitors in an undifferentiated state and prevent their terminal differentiation. Subsequently, mesenchymal signals were found to be critical for regulating pancreatic outgrowth and exocrine morphogenesis and cell differentiation, including FGF family members, epidermal growth factor, and members of the transforming growth factor (TGF) β/BMP superfamily (reviewed by Serup ). Currently, there is limited information on the endogenous mesenchymal signals required for human pancreas development; however, many of the mesenchymal signals identified in rodents are necessary for the stepwise differentiation of human embryonic stem cells into pancreatic lineages. ^, ^,

Disruption of mesenchymal signaling pathways often results in reciprocal changes in the endocrine versus exocrine cell fates. Mouse studies have identified a population of multipotent progenitor cells that can give rise to all pancreatic lineages in response to secreted morphogens. As pancreatic morphogenesis proceeds, the multipotent progenitor cells become allocated to specific tip and trunk domains within the pancreatic epithelium; cells within the trunk domain contribute to the duct and endocrine lineages, whereas the tip progenitors primarily give rise to the exocrine lineage (see Fig. 85.3 ). In humans, morphologic studies and limited marker analysis suggests that tip-trunk compartmentalization occurs in a similar manner between gestational weeks 7 and 14.

Extensive molecular analyses in mice have identified many additional transcriptional regulators of pancreas development. Shortly after bud evagination at embryonic day 9.5, Sox9 becomes induced in the Pdx1 / Ptf1a -expressing multipotent progenitor cell population. Between embryonic days 10.5 and 11.5, the multipotent progenitor cells acquire Nkx2-2 , Nkx6-1 , Hes1 , Hnf1b , Cpa1 , and Nkx6-2 expression. Subsequently, at embryonic day 12.5, the pancreatic precursor cells become spatially and molecularly diversified into exocrine and endocrine lineages. In response to signaling events that are not well understood, epithelial stratification initiates a series of morphogenetic events, including tubulogenesis and epithelial tubule remodeling that shape the mature functional branching organ with distinct trunk and tip domains. Concurrent with the morphologic changes, there is progressive restriction of transcription factor expression and corresponding lineage specification. Cells within the trunk domains maintain expression of Hnf1b , Nkx2.2, Nkx6-1 , and Nkx6-2 to give rise to the ductal and endocrine lineages, whereas the tip domain maintains expression of Ptf1a and Cpa1 to specify the exocrine lineage. Pdx1 and Sox9 continue to be expressed in all lineages. Reciprocal regulatory interactions between the Nkx6 factors and Ptf1α contribute to the delineation and maintenance of exocrine and endocrine lineages. Several immunohistochemical studies of human fetal tissue have demonstrated that many of these morphologic and gene expression events are conserved in the human pancreas, with the exception of NKX2-2, which is not detected until after endocrine cell specification. ^, ^, ^,

In mice, Notch signaling is also a key participant in pancreatic cell fate decisions. ^, Notch signaling is active throughout the multipotent progenitor cells as indicated by the expression of Hes1 , a major Notch target. Subsequent silencing of Notch signaling in the endocrine lineage leads to the down-regulation of Hes1 and the induction of the basic helix-loop-helix transcription factor Neurogenin3 (Neurog3). Neurog3 expression delineates the endocrine precursor population, and Neurog3 activity is required for all endocrine cell lineages. Successive cell fate decisions further diversify the Neurog3–expressing precursor pool by induction of a combinatorial hierarchy of transcription factors that promote a lineage-specific program. Whereas Notch is required for activation of Ptf1a and early commitment to the exocrine lineage, continuous activation of Notch signaling eventually inhibits exocrine development. ^, Notch signaling remains active in the centroacinar and terminal duct cells, which co-express Hes1 and Sox9 . In humans, the relative NOTCH1 , NEUROG3 , and HES1 expression domains are comparable to those in mice. ^, Furthermore, appropriate human endocrine cell development is dependent on functional NEUROG3 activity; human patients born with NEUROG3 mutations have variable defects in pancreas development, and many of these NEUROG3 alleles disrupt the differentiation of β cells from human pluripotent stem cells. ^,

Pancreatic Islet Cell Development and Function

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