DYNAMICS OF PANCREATIC DEVELOPMENT
Organogenesis of the pancreas is a highly coordinated process (Table 1). The first visible evidence of pancreas development in rodents is the formation of two independent thickenings, or “anlage,” each containing a few hundred cells, dorsally and ventrally, of the endodermal primitive gut tube at the 20- to 22-somite stage (Wessells and Cohen, 1967; Pictet and Rutter, 1972; Pictet et al., 1972). These regions are found immediately posterior to the anlagen of the stomach.
|Time point||Event||Morphological appearance||Gene expression|
|Gastrulation||Germ layer formation||Cell movement through the primitive streak generates mesoderm and endoderm.||Foxa2 in endoderm|
|6–10 somites||Determination, primary transition||Primitive gut as flattened sheet, no visible pancreas. Intimate contact of endoderm to the notochord. Signaling necessary. Ventral pancreas defined by absence of cardiogenic mesenchyme/septum transversum instruction||Isl1 expressed in dorsal mesenchyme.|
|10–20 somites||Embryo rotation||Closure of the gut tube. Formation of dorsal aorta between the gut tube and the notochord. Endoderm moves ventrally, displaced by aorta. No demarcation between anlage of stomach and pancreas. Mesodermal cells, “splanchnic mesoderm,” accumulate laterally on the gut tube.||Isl1 and Isl2 expression in mesoderm. Pdx1 activation (11–13 somites). HlxB9 expressed|
|20–25 somites||Budding||Thickening of dorsal/ventral walls of the gut, followed by epithelial evagination dorsally. Pancreatic mesoderm accumulates dorsally.||Nkx6.1 activation. Glucagon-positive cell clusters. Expression of Isl1 and Pax6in endocrine cells. Ngn3 expressed.|
|26–30 somites||Accumulation of mesoderm primarily on the left side of the gut generates asymmetry. The base of the dorsal evagination narrows. Ventral bud appears adjacent to liver bud.||Pitx2 involved in left/right handedness of primitive gut.|
|30+ somites||Continuous epithelial evagination. Branching morphogenesis initiates. Mitotic index highest in mesenchyme. Innervation commences (50 somites).||NeuroD, Ptf1a expressed. Epithelium strongly expressing E-cadherin and β-catenin.|
|E12 mouse||Visible branching morphogenesis||Latest time point for mesenchymal–epithelial separation.||Hnf6 homogeneously expressed in epithelium.|
|→ E14 (mouse)||Epithelial expansion||“Protodifferentiated” epithelium.||Nkx6.1, Pdx1, Nkx2.2 homogenously expressed in epithelium.|
|→ E15.5 (rat)||Very low-level presence of terminal markers (insulin, amylase, CPA, and others).||Pax6 and NeuroD speckled.|
|Fusion of dorsal and ventral pancreas.||High-level Notch1, 2, Hes1 expressed in epithelium.|
|Mesenchymal percentage high, but decreases gradually due to epithelial growth. Vascularization The expanding epithelium phase overlaps the onset of the secondary transition.|
|Appx. E13.5 (mouse)||Secondary transition||Terminal differentiation within β-cell and exocrine compartments.||> 100 fold activation of insulin and exocrine genes.|
|Appx. E15 (rat)||Appearance of zymogen granules. Scattered appearance of mature β-cells.||Pdx1, Nkx6.1, Nkx2.2 become β-cell restricted.|
|Pax4 activated. Ngn3 expression peaks.|
|E15–E19, prenatal (mouse)||Isletogenesis||Endocrine cells aggregate in islets of Langerhans. Exocrine acinary ultrastructure attained. Functional organ at birth.||Delta-cells appear. (App. E15) PP cells appear.|
|P1–P30||Growth||Tissue mass increase. High percentage of endocrine cells in the early postnatal phase is overtaken by massive growth of the exocrine mass, which condenses to form compact acini.||Trypsin and CPB activated (P1).|
The onset of pancreagenesis is known as the “primary transition,” referring to the change in shape of the pancreatic domains of the gut and the attained pancreatic cellular state. Wessells and colleagues demonstrated that the period of pancreatic determination occurs between the 6- and 10-somite stages in the rat (corresponding to embryonic day [E] 8.0 in the mouse; Wessells and Cohen, 1967; Wessells and Evans, 1968). More recent studies have revealed that the dorsal and ventral programs of the pancreas are not completely identical and slightly asynchronous, due to the two independent endodermal domains receiving distinct signals from their surrounding tissues.
The dorsal pancreatic bud develops in close proximity to the notochord. This proximity is essential for dorsal bud development. Slightly later, as the notochord is displaced by the dorsolateral splanchnic mesenchyme, which forms the dorsal aorta in between the dorsal epithelium and the notochord, a new set of signals are exchanged between the aorta and the pancreas. Recent studies of the molecular mechanisms that play a role in these processes have revealed the involvement of several morphogenetic signaling systems (including fibroblast growth factor [FGF], transforming growth factor-beta [TGFβ], vascular endothelial growth factor [VEGF], and hedgehog-type ligands; Kim et al., 1997; Hebrok et al., 1998, 2000; Wells and Melton, 2000). The early morphogenetic signals have been reviewed excellently previously (Melton et al., 1997; Wells and Melton, 1999; Grapin-Botton and Melton, 2000).
The ventral bud of the pancreas develops in close connection to the liver and bile duct epithelium that forms on the ventral face of the gut. The ventral pancreas originally develops as two independent ventral foregut endodermal regions, laterally positioned, which are brought together at the time of gut tube closing. Ventral pancreatic development is closely connected to organogenesis of the liver, with the latter being specified due to its proximity instructive signaling from the cardiac mesoderm. Concomitantly, the ventral pancreatic program is initiated due to the absence of this instructive signaling. The ventral pancreatic fate program appears to be a default state, because in the absence of cardiac induction cells of the prospective liver domain attain a ventral pancreatic fate (Zaret, 2000, 2001; Deutsch et al., 2001). Even in adult mouse pancreas, the plasticity between liver and pancreas fates can be observed (Shen et al., 2000) and often occurs naturally (Grompe, 2003). Such observations of plasticity between endodermal fates are not unique. Flexibility also exists between the pancreas and stomach fates (Hald et al., 2003), as well as between pancreatic vs. duodenal fate (Apelqvist et al., 1997; Kawaguchi et al., 2002). Such observations clearly point to the existence of shared genetic components amongst these endodermal derivatives.
After its initial specification, the epithelial thickening comprising the nascent pancreas changes over the next day into proper epithelial evaginations. These form perpendicular to the gut wall, destined to form the duodenum (Fig. 1). The epithelium is of a multilayered stratified type consisting of cells with a high nuclear/cytoplasmic ratio. This composition is very different from the later single-layered epithelium of the mature pancreatic ducts. At this stage, mesodermal cells (pancreatic mesenchyme) tightly associate with the pancreatic epithelium. They guide its development mainly through proliferative signaling (Wessells and Cohen, 1967). In both mouse and rat, the pancreatic epithelium expands markedly in the following days, during which the stratified epithelial nature is maintained, although a gradual thinning occurs and epithelial lobulations become pronounced.
It is generally recognized that the pancreas undergoes a process of branching morphogenesis, while the epithelial expansion ensues. However, as different mechanistic solutions exist to coordinate such a process, care should be taken when using this nomenclature. As recently pointed out by Hogan and Kolodziej (2002), tubulogenesis differs structurally between organs generally recognized as undergoing branching morphogenesis. In the lung, a continuous expansion and folding of the epithelial layers is observed and this process is governed by mesenchymal–epithelial cross-talk. This particular process is of the classic branching morphogenesis type (Fig. 2A). In contrast, in both pancreas and salivary glands the formation of a ductal tree occurs through a process of intraepithelial lumen formation, wherein multiple microlumens eventually coalesce to form continuous lumens around which the ductular network is established (Fig. 2B). This is the first time point that cells having a typical basal–apical polarity signifying a cuboidal/rectangular epithelial are observed in the pancreas. Subsets of the endodermal cell population thereby form a ductal tree that, in the mature state, drains exocrine secretory products. Probably closely linked to the morphologic changes thus imposed, more differentiated characteristics appear, such as expression of ductal marker genes (e.g., CFTR, carbonic anhydrase II, mucins, and increased expression of certain cytokeratins; Githens, 1988). At present, we have only a descriptive understanding of pancreatic ductular development—no data have been derived that explain how the given cellular subsets of the pancreatic endoderm are allocated to a ductal fate, nor do we understand the mechanism underlying pancreatic tubulogenesis.
Our current appreciation of pancreatic development is strongly founded upon electron microscopic analyses addressing the presence of individual cell types in development (Pictet et al., 1972). From the presence of secretory granules, clearly distinguishable as being of β-cell, α-cell, and exocrine types, it was demonstrated that mature β-cells and exocrine cells were not detectable before embryonic day 15 in the rat (E15, corresponding to E13.5 in mice). However, glucagon-producing cells were detected at the early somite stages of development, concomitant with the budding process. On the basis of these data, Rutter and colleagues proposed a model of pancreatic development describing two major transitions in time. During the “primary transition,” cells at the appropriate regions of the gut become destined to form pancreas. These acquire molecular identities, which may be referred to as the “the pancreatic state” as described by Slack (1995). Only a few cells containing α-cell–like granules differentiate, and the remaining epithelial cells are characterized as being “protodifferentiated” (Pictet et al., 1972). The term “protodifferentiated” was used to reflect the presence of low levels of pancreas-specific terminal gene products, such as insulin and amylase in pancreatic extracts. This terminology may still be well suited to describe cells in “the pancreatic state.” At the time, no knowledge was available to support concepts of cell intrinsic regulators that might confer such identity.
During “the secondary transition”—a relatively short period between E13.5 and E15.5 in mouse (Fig. 3) —protein concentrations of β-cell and exocrine products increase between 100- and 1,000-fold (Han et al., 1986). A concomitant increase is observed for insulin, amylase, carboxypeptidase A, lipase, and chymotrypsin. At this time point, the structure of the adult organ begins to be recognizable. These descriptions of the primary and secondary transitions have been substantiated by a range of alternative techniques, including histologic approaches (immunofluorescence, in situ hybridization), and molecular techniques (Northern blotting, reverse transcriptase-polymerase chain reaction, DNA chip analyses). After the secondary transition, few cells of the pancreas progenitor type remain and expansion occurs by growth of fully differentiated cell types. Exocrine cells are strongly proliferating after their differentiation, and endocrine cells show strong mitotic activity closer to birth. In the interim, the endocrine cells aggregate by migration into clusters in a process that may be termed “isletogenesis” (Fig. 4).
Lineage Determination During Pancreagenesis
The appearance of the glucagon-containing cells within the primitive buds is the first evidence of cytodifferentiation of pancreatic cells. It had been suggested that these are precursors for all cells of the endocrine cell lineage. This notion dominated the pancreatic development field for almost two decades. However, lineage relation analyses have proven that this is not true. Herrera and colleagues (1994) showed, by targeting the diphtheria toxin A (Dip.ToxA) chain to α- or β-cells by using the glucagon and insulin promoters, respectively, that insulin and glucagon-producing cells were not related through a coexpressing precursor cell. Insulin promoter–driven Dip.ToxA only eliminated β-cells, and the glucagon promoter–driven Dip.ToxA selectively destroyed α-cells. Due to its inconsistency with the then-current notion, the study was not generally accepted, as it could be argued that the activity of the toxin was not sufficiently rapid, or effective, to kill the transient precursor cell.
Herrera later performed a lineage analysis using the Cre-Lox method of labeling a cell irreversibly. Asking whether an insulin cell ever expressed glucagon during an earlier stage, he crossed glucagon-promoter Cre-recombinase mice with a reporter mouse carrying a silenced loxP-STOP-loxP-flanked growth hormone allele. In the presence of the Cre-recombinase, the reporter would be activated, and in the presence of growth hormone/insulin, double-positive cells could be analyzed by histology. These were not found, showing that insulin-cells do not develop through a glucagon-expressing stage (Herrera, 2000). As the reciprocal experiment also showed that glucagon-cells do not develop through an insulin-expressing stage, it was concluded that these cell types develop independently. Immunohistochemical data, based on expression of transcription factors rather than the pancreatic hormones, are consistent with these results (Jensen et al., 2000a). Nevertheless, a common origin of endocrine cells does exist at a non–hormone-expressing precursor level where both α- and β-cells develop from PDX1-positive cells. This finding was demonstrated by using Pdx1-promoter CRE-mice, which generated an active reporter gene in both α- and β-cells (Herrera, 2000). Extending these observations, Gu and colleagues showed that all endodermally derived cells of the pancreas—endocrine, exocrine, and ducts—were originating from Pdx1-expressing precursors (Gu et al., 2002). Furthermore, by taking advantage of tamoxifen-inducible recombination by generating mice expressing the CreERT protein (a Cre-recombinase:estrogen receptor ligand binding domain [ER-LBD] chimera, with three point substitutions in the ER-LBD rendering the nuclear receptor responsive to tamoxifen) under control of the Pdx1 and Ngn3 promoters, the window of recombination in Pdx1-expressing cells could be controlled. Of interest, cells of the ductal lineage appeared to be specified within a short period from E9.5 to E12.5 in the mouse, as only during this time did tamoxifen-induced recombination lead to labeling of mature duct cells (Gu et al., 2002). Such temporal restriction was not observed for exocrine and endocrine cells. Contrasting the common Pdx1-expressing ancestry of all pancreatic cells, Gu observed that cells expressing Ngn3-Cre were almost exclusively of endocrine types, an observation in agreement with other studies (Gradwohl et al., 2000; Jensen et al., 2000a; Schwitzgebel et al., 2000). Finally, Kawaguchi et al., by performing a modified knock-out approach of the Ptf1a gene in which the Cre-recombinase was inserted into the locus in the process, revealed that pancreatic exocrine cells arise from Ptf1a-expressing precursors—but also discovered that a high percentage of all pancreatic cell types (except for the earliest glucagon-expressing cells) could be traced by this method, showing that Ptf1a, like Pdx1, is expressed within undifferentiated pancreatic precursors (Kawaguchi et al., 2002). Together, these studies demonstrate that the mature pancreatic cells develop through a common precursor cell stage, which express the genes Pdx1 and Ptf1a—and that differentiation toward the endocrine lineages occur through a transient Ngn3 expressing precursor cell, which is not shared with nonendocrine lineages.
In addition to the above genetic studies, tracing of cells can also be performed by marking individual cells at one given stage in development, followed by investigations of the phenotype of the progeny. Fishman and Melton recently obtained further evidence for the existence of a common endo/exocrine precursor cell using a replication-deficient retroviral labeling of pancreatic explants (Fishman and Melton, 2002). This study complements the Cre-LoxP methods and highlights the usefulness of such additional approaches, which has not been exploited to any large extent in the developing pancreas. Such experiments, as well as dedicated clonogenic assays should be capable of revealing the level of lineage relationships, lineage restrictions, and timing of developmental competence in the embryonic pancreas.