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An Illustrated Review of Early Pancreas Development in the Mouse

Hagedorn Research Institute, Department of Developmental Biology, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark

Correspondence: Address all correspondence to: Jacob Hecksher-Sørensen, Hagedorn Research Institute, Department of Developmental Biology, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. E-mail: jhes{at}hagedorn.dk. Address reprint requests to: Mette Christine Jørgensen, Department of Developmental Biology, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. E-mail: mecj{at}hagedorn.dk

	Abstract

Top Abstract I. Introduction II. From Endoderm to... III. Pancreatic Endocrine and... IV. Transcription Factor... V. Pancreatic Endocrine... VI. Pancreatic Endocrine Cell... VII. Summary and Future... Note Added in Proof References

 
Pancreas morphogenesis and cell differentiation are highly conserved among vertebrates during fetal development. The pancreas develops through simple budlike structures on the primitive gut tube to a highly branched organ containing many specialized cell types. This review presents an overview of key molecular components and important signaling sources illustrated by an extensive three-dimensional (3D) imaging of the developing mouse pancreas at single cell resolution. The 3D documentation covers the time window between embryonic days 8.5 and 14.5 in which all the pancreatic cell types become specified and therefore includes gene expression patterns of pancreatic endocrine hormones, exocrine gene products, and essential transcription factors. The 3D perspective provides valuable insight into how a complex organ like the pancreas is formed and a perception of ventral and dorsal pancreatic growth that is otherwise difficult to uncover. We further discuss how this global analysis of the developing pancreas confirms and extends previous studies, and we envisage that this type of analysis can be instrumental for evaluating mutant phenotypes in the future.

I. Introduction

II. From Endoderm to Pancreas—e8.5 to e10.5

A. Patterning of the endoderm

B. Specification of the pancreatic endoderm

C. Expansion of the pancreas progenitor pool

III. Pancreatic Endocrine and Exocrine Cell Compartments

A. Growth and branching of the pancreas

B. The peptide hormones

C. The exocrine pancreas

IV. Transcription Factor Signature of the Multipotent Pancreatic Progenitor Cells

A. Pdx1

B. Hlxb9

C. Ptf1a

D. Nkx6-1 and Nkx2-2

V. Pancreatic Endocrine Progenitor Cell Determination

A. Neurog3

B. Nkx2-2

C. Pax6

D. Pou3f4

VI. Pancreatic Endocrine Cell Fate Determination

VII. Summary and Future Aspects

	I. Introduction

Top Abstract I. Introduction II. From Endoderm to... III. Pancreatic Endocrine and... IV. Transcription Factor... V. Pancreatic Endocrine... VI. Pancreatic Endocrine Cell... VII. Summary and Future... Note Added in Proof References

 
NEW INSIGHTS FROM basic and clinical research suggest that the common denominator of all forms of diabetes is the lack of an adequate functional ß-cell mass (an absolute lack in type 1 diabetes vs. a relative lack of ß-cells in type 2 diabetes) and thereby insulin insufficiency leading to hyperglycemia (1, 2). Reconstitution of a functional ß-cell mass would thus predict the reestablishment of euglycemia as well as protection against vascular complications. Proof of principle that cell therapy of type 1 diabetes can restore euglycemia was provided by the "Edmonton protocol" (transplantation of organ donor islets of Langerhans) (3) and further validated by Pipeleers and co-workers (4). Organ donor material is scarce, and alternative sources for therapeutic ß-cells generated from stem cells represent a highly active area of research (reviewed in Ref. 5) that requires a deep and thorough understanding of pancreas developmental biology with the focus on ß-cell ontogeny.

The tremendous advance in developmental biology that has occurred since the first comprehensive review by Rutter et al. (6), who introduced the concepts of the primary and the secondary transition, has greatly expanded our knowledge of pancreatic development. This detailed description of how the morphological events of pancreas formation correlate with cytodifferentiation (7) has since formed the basis for the work of contemporary pancreas biologists. Then, loss- and gain-of-function studies in various animal models, fate mapping and lineage tracing studies, cell sorting, and array techniques have all contributed to a better understanding of pancreatic specification, growth, and differentiation. The earliest stages of pancreas organogenesis depend on signaling interactions with surrounding tissues, and the local environment in which the pancreas develops is highly dynamic regarding the relative positioning of the different tissues. Initially, the pancreas primordia are relatively simple structures containing the common pancreatic progenitor cells, but the complexity increases with time due to the appearance of numerous differentiated cell types, gut rotation, and branching morphogenesis. Before organ specification, the formation of definitive endoderm during the gastrulation process is controlled by multiple signaling factors and morphogens where the TGFß family member nodal seems to be a key molecule in vertebrates (8, 9). By mimicking the signaling events that the endocrine progenitor cells are exposed to during development, it has recently proven possible to direct the differentiation of human embryonic stem (ES) cells into endoderm (10, 11) and subsequently differentiate the endoderm into hormone-expressing endocrine cells (12). These studies have emphasized the requirement for understanding the precise timing of signaling events taking place during the various stages of the ß-cell differentiation.

The last decades have also witnessed technological advances in microscopy and computer power making it possible to generate reconstructions of tissue in three dimensions (3D). This has already proven valuable when applied to whole organisms like zebrafish and fruit fly (13, 14) and also for whole organ analysis (15, 16). To illustrate important morphological events and the temporal and spatial changes in gene expression patterns during pancreas development, we provide 3D images obtained with laser scanning microscopy of intact mouse pancreata (17). We find that this technology offers a high level of detail in the description of spatial and temporal events during pancreas development.

Here, we review the initiation of organ growth and subsequent expansion along with the expression profiles of molecular markers at the early stages of pancreas development during mouse embryogenesis from embryonic day 8.5 (e8.5) to e14.5. In short, each individual image of the five composite figure panels represents one intact fetus or dissected pancreas that has been double or triple stained for specific markers by indirect immunofluorescence. The specimen is optically sectioned, and the signal is stored individually for each color channel for each section. The pictures are generated by overlay of the colors followed by collapsing all sections into one. To appreciate the details in the figures we refer to the supplementary files (published as supplemental data on The Endocrine Society’s Journals Online web site at http://edrv.endojournals.org) that provide movies comprising all the optical sections of each image stack with single cell resolution. The raw data sets are available on request. Here, it is possible to separate the color channels and generate rotating 3D reconstructions for each individual figure image. We have used a number of molecular markers expressed during early pancreas development to visualize the following: 1) tissue-tissue interactions; 2) morphogenetic events in time; 3) coexpression of marker genes as well as single cell distribution; and 4) the perturbed early gene expression in the two mouse mutants, Hes1^{–/ –} and Neurog3^{–/ –}. Together, this provides a detailed systematic review of developmental stages and molecular markers in pancreas formation.

	II. From Endoderm to Pancreas—from e8.5 to e10.5

Top Abstract I. Introduction II. From Endoderm to... III. Pancreatic Endocrine and... IV. Transcription Factor... V. Pancreatic Endocrine... VI. Pancreatic Endocrine Cell... VII. Summary and Future... Note Added in Proof References

 
A number of classical studies describe the morphological aspects of early pancreas formation, which in mouse becomes evident at e8.75–e9.0 immediately after closure of the anterior endoderm. Dorsally, a thickening of the endoderm appears opposite of the hepatic outgrowth on the ventral side. Over the next day from e9.0 to e10.5, the dorsal pancreatic epithelium continues to thicken and form a proper bud, and the base of the diverticulum becomes progressively constricted to form a stalk (7, 18, 19). Isolated pancreatic endoderm can grow and differentiate in vitro and is committed to form pancreatic structures in organ culture experiments before morphogenesis can be observed (18). However, growth and cytodifferentiation is dependent on the presence of pancreatic mesenchyme at the earliest stages, whereas later development can be promoted by heterologous mesenchymal sources (18, 20). That the mesenchyme produces and secretes diffusible growth-promoting factors was elegantly shown by coculture experiments where pancreas development could proceed although the epithelium was separated from the mesenchyme by a filter membrane (20).

A. Patterning of the endoderm
In both mouse and chicken embryos, the formation of the gut tube involves the conversion of a flat sheet of endoderm to a primitive gut tube structure (21). A crescent-shaped fold appears at the anterior end of the embryo simultaneously with the beginning of somitogenesis. This fold, called the anterior intestinal portal, progresses posteriorly along the body axis and leaves behind the anterior part of the primitive gut tube. Later, a similar fold, the caudal intestinal portal, forms at the posterior end of the embryo, and it moves anteriorly to form the posterior parts of the primitive gut until the two folds meet at the yolk stalk (22, 23). A number of gene products appear to be instrumental for the closure of the gut tube to occur. Thus, the GATA-binding transcription factor 4, the proprotein convertase Furin, the matrix metallopeptidase 2, and several of the bone morphogenic proteins (BMPs) have all been shown to be important factors during early stages of gut tube formation (24, 25, 26, 27, 28).

Concurrent with the formation of the intestinal portals, the gut tube is also patterned in an anterior to posterior manner, and discrete regions of the endoderm can be identified using fate mapping and molecular markers. Midline endoderm becomes dorsal gut tube, and the lateral endoderm underlying the lateral plate mesoderm contributes to lateral and ventral gut tube (see Refs. 22 and 29 ; reviewed in Refs. 9, 21 and 30). Regarding pancreas development, the most extensive endodermal fate maps have been obtained in chick embryos showing that the dorsal pancreas arises from the midline endoderm between the third and ninth somite pair (31, 32). The ventral pancreas develops from two lateral areas of endoderm at the same level (23, 32). Here, both the midline and the lateral areas of endoderm express the pancreatic transcription factors Pdx1, Nkx6-1, Nkx6-2, and Nkx2-2 before actual closure of the gut tube and before any anatomical characteristics of pancreatic tissue are visible (33).

Patterning of the gut tube depends on signals from the adjacent lateral plate mesoderm, which secretes soluble factors like fibroblast growth factors (FGFs), BMPs, activin, and retinoic acid (RA) (32, 34). It has been shown that RA-mediated signaling is required for dorsal pancreas specification (35, 36, 37, 38, 39) and that the RA from the mesoderm signals directly to the pancreatic epithelium (39, 40, 41).

In the decades following the early morphological studies, a large number of molecular markers for pancreatic cell types and stages have been described. First among these was Pdx1 which can identify pancreas specification before any visible changes in morphology (Figs. 1 and 2) (42, 43, 44). Although Pdx1 is recognized as the earliest and most specific gene expressed in the pancreas primordia (44), other factors such as Hlxb9 (Hb9), Hhex (Hex), Onecut1 (HNF6), Tcf2 (vHNF1, HNF1ß), and Foxa2 (HNF3ß) are known to precede Pdx1 expression in mice (43, 45, 46, 47, 48, 49, 50, 51). However, these genes are more widely expressed and therefore cannot be used as specific markers of the pancreas. At e8.5 (8–10 somites) in the mouse, two ventral Pdx1 domains appear just before closure of the endoderm (Fig. 2A, inset), illustrating that expression of Pdx1 in the ventral domain precedes that of the dorsal domain (43) (Fig. 2, A and B). Low levels of Pdx1 immunoreactivity can also be observed in the visceral endoderm at e8.5 (data not shown), confirming a previous study detecting Pdx1 by RT-PCR in e7.5 and e8.5 visceral yolk sac (52) and recent microarray data also detecting Pdx1 mRNA in e8.25 visceral endoderm (53). The first dorsal Pdx1 expression appears at the 10 somite stage between e8.5 and e8.75 when the embryo turns and the endoderm is still closely associated with the notochord (Figs. 1, B and F, and 2, C and D).

View larger version (50K): [in this window] [in a new window]   FIG. 1. Positioning of notochord and dorsal aorta during pancreas specification. Collapsed image stacks of whole mount immunohistochemical detection: A–D, Pdx1 in the pancreatic epithelium, T in the notochord, and Cdh1 in epithelial cells in general; and E–H, Pdx1 in the pancreatic epithelium, Pecam1 in endothelial cells, and Foxa2 staining all endoderm and neural tube floor plate in e8.5–e9.0 wild-type mouse embryos. A and E, Low magnification overview pictures of the e9.0 embryos shown in panels C and G. B, Between e8.5 and e8.75, the notochord lies in close proximity to the dorsal pancreatic epithelium. C and D, At e9.0, the notochord and the pancreatic epithelium become separated, and the distance between them increases as development proceeds. F, Between e8.5 and e8.75, there are two lateral aortas along the sides of the notochord. G and H, At e9.0, the two aortas fuse between the notochord and the dorsal pancreatic epithelium. In all panels, anterior is to the right, posterior is to the left, dorsal is above, and ventral is below.

View larger version (67K): [in this window] [in a new window]   FIG. 2. Pancreas morphogenesis from e.8.5 to e14.5. Whole mount immunohistochemical detection of Pdx1, Nkx6-1, and Cdh1 in wild-type e8.5 to e10.5 whole embryos (A–L) and wild-type e11.5 to e14.5 dissected pancreas and stomach (M–T). In all panels, anterior is to the right, posterior is to the left, dorsal is above, and ventral is below. Panels B, D, F, H, J, and L are image stacks of high-magnification scans of the pancreas regions of the image stacks shown in panels A, C, E, G, I, and K, respectively. Panels N, P, R, and T are high-magnification optical sections from the image stacks shown in panels M, O, Q, and S, respectively. A and B, Ventral Pdx1 expression can first be detected at e8.5 before the embryos turn. A, inset, Another e8.5 embryo allowing a more ventral view exposes two ventral Pdx1 domains. C and D, At e8.75, the dorsal expression of Pdx1 can be detected and a few cells transiently expressing Nkx6-1 can be observed in the ventral domain (D, inset). E–L, From e9.0 to e10.5, the structures of both the dorsal and the ventral pancreas become defined. E and F, Nkx6-1 expression can first be detected in the dorsal pancreas from e9.0. K and L, The ventral expression of Nkx6-1 reappears at e10.5. L, Pdx1 and Nkx6-1 are coexpressed at high levels at e10.5 in the epithelia of both pancreatic buds. N and M, At e11.5, the expression patterns of Pdx1 and Nkx6-1 start to deviate, and Nkx6-1 becomes more restricted to the central part of the epithelium and disappears in cells adjacent to the mesoderm. O, At e12.5, the dorsal and ventral pancreas parts fuse to become one interconnected organ. O, Q, S, Substantial growth and branching of both dorsal and ventral pancreas take place from e12.5 to e14.5 (Q–T; only dorsal pancreas parts are visualized). P, R, T, At these stages, Nkx6-1 becomes completely excluded from the branching tips, which are still positive for Pdx1 expression (highlighted by inset and arrows). Q–S, From e13.5, scattered cells expressing high levels of Nkx6-1 and/or Pdx1 can be detected throughout the central part of the pancreas (see also supplemental data because these cells are better observed on the optical sections). U–Y, Schematic drawings showing the Pdx1-expressing area of the endoderm at e9.5 (U), e10.5 (V), e11.5 (X), and e12.5 (Y). A, Anterior; P, posterior; dp, dorsal pancreas; vp, ventral pancreas; li, liver; du, duodenum; st, stomach.

View larger version (59K): [in this window] [in a new window]   FIG. 4. Expression of pancreatic transcription factors. Collapsed image stacks of wild-type whole embryos (e9.0–e10.5) or dissected pancreata (e11.5 and e12.5). To visualize the pancreatic epithelium, all embryos were stained for Pdx1 and only the dorsal pancreata are shown at e12.5. In all panels, anterior is to the right, posterior is to the left, dorsal is above, and ventral is below. A1, HBlx9 is expressed broadly in the dorsal endoderm before the onset of Pdx1 expression (inset; dorsal Hlxb9 expression at e8.5 is shown where only a few dorsal Pdx1 cells have appeared). Also, Hlxb9 expression is seen in the notochord and the neural tube. A1–A4, Although endodermal Hblx9 expression gradually becomes excluded from the duodenum, it remains expressed throughout the dorsal pancreas, and from e9.5 it can also be detected in the ventral pancreas (A3, inset; arrow shows ventral Hlxb9 expression at e9.5 without the overlapping Pdx1 staining). A5 and A6, At e11.5 and e12.5, Hlxb9 becomes down-regulated and disappears from the dorsal and the ventral pancreas endoderm but can still be detected in scattered cells in the central part of the pancreas epithelium. B1 and B2, Ptf1a can be detected simultaneously in both the dorsal and ventral pancreas at e9.25. Ptf1a is known to be expressed before e9.25, but the antibody used here fails to detect any expression at e9.0. B2–B5, Until stage e11.5, Ptf1a is expressed throughout the dorsal and ventral pancreas endoderm. B6, At e12.5, Ptf1a becomes completely restricted to the tips of the branching epithelium. C1 and C2, The proendocrine gene Neurog3 can be detected robustly at e9.0. Most of the Neurog3-positive cells reside within the Pdx1 expression (Continued) domain, but a number of cells can also be observed in the endoderm between the two pancreas domains. This is also true at e9.25. C3–C6, From e9.5 to e12.5, Neurog3 becomes restricted to the pancreas endoderm. (At later stages not described here, Neurog3 will be expressed in the duodenum.) C3, The first ventral Neurog3 expression can be detected at e9.5. C6, At e12.5, Neurog3 is expressed in a scattered pattern in the central part of the epithelium. D1–D6, Pancreatic Nkx2-2 expression marks two populations of cells. It is expressed at high levels in the endocrine lineage and at low levels throughout the pancreatic endoderm (see also Fig. 6C). It is difficult to show both expression patterns in the same image because recording of the low signal results in overexposure of the high-level expressing cells. Nkx2-2 expression first appears at e8.75 within the dorsal Pdx1 expression domain (D1, inset) and in the ventral pancreas from e9.5 (D3, inset; arrow shows ventral Nkx2-2 expression at e9.5 without the overlapping Pdx1 staining). The low expression can be detected throughout the pancreas endoderm until e12.5, when it becomes restricted to the central part of the epithelium and the endocrine cell clusters (D1–D6; also see supplemental data). The newly born endocrine cells that express high levels of Nkx2-2 can be detected in a scattered pattern at all stages described here. At e9.0 and e9.25, some of these cells appear outside the Pdx1 expression domain (D1 and D2), but later they are solely found within the pancreas area (D3–D6). In the ventral pancreas, high-level Nkx2-2 expression can be detected from e9.5 (D3). Note also the Nkx2-2 expression in the ventral neural tube (D1). E1–E6, The expression of Pax6 is slightly delayed compared with Neurog3 and Nkx2-2 and can first be detected at e9.25 in the dorsal bud (E2) and at e10.5 in the ventral bud (E4) and has not been observed outside the pancreas domains. E5 and E6, At e11.5 and e12.5, many Pax6-expressing cells appear in clusters as observed for the hormones (also see Fig. 3). F1–F5, The pattern of Pou3f4 expression from e9.0 to e11.5 is similar to the pattern seen for the high-level Nkx2-2-expressing cells, except that Pou3f4 nuclei can be found continuously outside the pancreas domains. F6, At e12.5 the expression of Pou3f4 can be divided into low-level expressing endocrine cells located in clusters and scattered high-level-expressing cells. Although the number of cells expressing high levels of Pou3f4 has been comparable to those of Neurog3 and Nkx2-2 at the earlier stages (compare C1–C5 and D1–D5 with F1–F5), it appears relatively lower at e12.5 (compare C6 and D6 with F6).

FGF10 is expressed in the mesenchyme, and FGF10-deficient mice exhibit severe growth retardation of the pancreas (69), whereas ectopic FGF10 expression controlled by the Pdx1 promoter results in sustained proliferation at the expense of differentiation (70, 71). Apparently, the effect of FGF10 signaling on the epithelium is at least partly mediated by Notch pathway activation (70, 71, 72), which is known to inhibit both endocrine and exocrine differentiation. Mice deficient in various Notch signaling components all show accelerated differentiation at the expense of proliferation, resulting in pancreas hypoplasia (73, 74, 75, 76). Conversely, overexpression of a constitutively activated form of Notch1 in the pancreas epithelium prevents endocrine and exocrine differentiation (77, 78, 79, 80).

Several components of the TGFß signaling family are also important in organizing and regulating the growth and relative proportions of the endocrine and exocrine compartments in the pancreas. Both mesenchymal Follistatin and the expression of growth differentiation factor 11 in the pancreatic epithelium promote growth of the exocrine tissue (81, 82, 83). On the other hand, inactivation of the activin receptors IIA and B, the type II receptors of e.g. activins, BMPs, and growth differentiation factor 11, has severe effects on endocrine development resulting in islet hypoplasia (84).

Correct specification of the pancreatic epithelium is an additional prerequisite for pancreas morphogenesis, and the epithelial transcription factor mutants for Pdx1, Ptf1a, and Hlxb9 all result in impaired pancreas formation (43, 45, 85, 86, 87, 88). It has been shown that the lack of Pdx1 makes the pancreatic epithelium unable to respond to the mesenchymal growth-promoting signals (42), and the lack of Ptf1a results in compromised exocrine as well as endocrine cell formation and redirection of the fate of the pancreatic progenitor cells to become duodenal epithelium (88, 89). Moreover, transgenic mice with Pdx1 expressed under control of the Ptf1a promoter can restore pancreas formation when crossed onto the Pdx1 null background (88), demonstrating that Ptf1a expression is independent of Pdx1. RT-PCR combined with microarray-based gene expression profile analyses of individually isolated e10.5 dorsal pancreatic cells have also revealed that Pdx1, Ptf1a, Nkx6-1, Nkx2-2, and to a large extent Hlxb9 are coexpressed in most of the epithelial pancreatic progenitor cells (90). Recent work identifies another important transcription factor, Sox9, in the pancreatic progenitor cells (91). Sox9-deficient mice display severe pancreas hypoplasia and very few scattered endocrine cells, demonstrating that Sox9 activity is required during development for pancreatic growth and endocrine cell differentiation (91).

In summary, the developmental progression at the stages from e8.5 to e10.5 defines the pancreas as an organ. In this time window, the transcription factor combinatorial code, which distinguishes the pancreatic cells from any other cell type in the embryo, becomes consolidated. Coexpression of Pdx1, Hlxb9, Ptf1a, Nkx6-1, and Nkx2-2 defines the common pancreatic progenitor cells in the epithelium (Fig. 2, A–L, and Fig. 4, A1–A4, B1–B4, and D1–D4) together with Nkx6-2 and Sox9 (33, 91), and an intricate unsolved interplay between different signaling pathways in the pancreatic mesenchyme is essential for growth. Interestingly, recent work suggests that the final size of the pancreas is determined by the original number of progenitor cells present already at this early stage (92).

	III. Pancreatic Endocrine and Exocrine Cell Compartments

Top Abstract I. Introduction II. From Endoderm to... III. Pancreatic Endocrine and... IV. Transcription Factor... V. Pancreatic Endocrine... VI. Pancreatic Endocrine Cell... VII. Summary and Future... Note Added in Proof References

 
The mature pancreas is a bifunctional organ primarily consisting of exocrine tissue organized in acini that secrete zymogens for digestive purposes and the ductal scaffold that drains the fluid from the acinar compartment. The pancreatic duct cells also serve an exocrine function as they secrete bicarbonate for neutralization of stomach acid in the duodenum. The islets of Langerhans, which harbor the different endocrine cell types, can be found embedded within the exocrine tissue (61).

The process of pancreatic cytodifferentiation and related ultrastructural characteristics have been extensively studied primarily in the rat (7, 93). The developmental state of the exocrine cells was followed closely by enzyme assays for a number of exocrine protein products (6), and the levels of the endocrine hormones insulin and glucagon were followed similarly during development by the use of immunoassays (94, 95). The resulting profiles of exocrine enzyme activity and endocrine hormone content during pancreatic development led to the proposal of a triphasic model of key regulatory transition events (6). The primary regulatory transition (e9.5–e11.5, corresponding to e8.5–e10.5 in the mouse) is related to organ determination and is defined as the conversion of predifferentiated cells to a protodifferentiated state where low levels of pancreas-specific proteins are present. The secondary regulatory transition (e14.5–e19.5, corresponding to e13.5–e16.5 in the mouse) is the conversion of protodifferentiated tissue to fully differentiated cells characterized by an immense increase in pancreas-specific protein synthesis and by the loss of proliferative capacity. The third regulatory transition occurs after birth and denotes the adaptation to dietary regulated synthesis and secretion of pancreas-specific proteins (6). It has been reported that despite the delayed morphological appearance of the ventral compared with the dorsal pancreas bud, they develop simultaneously regarding exocrine differentiation. However, they differ significantly in the content and temporal expression of glucagon (19).

It appears that different culture conditions of in vitro-cultured pancreatic epithelium will promote the development of different pancreatic cell types (96). Mesenchyme-depleted mouse e11.5 pancreatic epithelium will generate duct cells when cultured in a basement membrane rich matrigel and endocrine cells when grown under the kidney capsule, whereas exocrine cells will only form in the presence of mesenchyme (96). However, another study with in vitro-cultured e12.5 rat pancreas shows that endocrine cells can develop and mature to form islet structures when the mesenchyme is removed from the epithelium, whereas the exocrine compartment needs the presence of mesenchyme to develop correctly (83). Recently, it has been demonstrated that the mesenchyme enhances the proliferation of the pancreas progenitor cells but later has an inhibitory effect on endocrine differentiation (97). The resulting delay in endocrine differentiation is important for the generation of sufficient ß-cells, and it is shown that the function of sulfated proteoglycans, which are localized in the mesenchyme and in the epithelial basement membrane, is required to mediate the inhibition (98). Moreover, glucose seems to positively regulate endocrine differentiation by stimulating the expression of the basic helix-loop-helix (bHLH) transcription factor Neurod1, whereas the exocrine tissue is unaffected by changes in glucose concentration levels (99). Other explant culture experiments also suggest a role for RA signaling in regulation of endocrine and exocrine differentiation (100, 101).

A. Growth and branching of the pancreas
After e10.5, the pancreatic tissue expands rapidly from a compact structure of tightly packed cells and grows severalfold in size, forming a clearly branched structure within a few days (Fig. 2, M–T). Simultaneously, the epithelial expression patterns of Pdx1 and Nkx6-1 start to deviate, and cells appear at the periphery that express Pdx1 but not Nkx6-1 (Fig. 2, N and P). This pattern becomes more evident at e13.5 and e14.5, where Pdx1 alone marks the forming acini, whereas Nkx6-1 becomes restricted to the central part of the epithelium (Fig. 2, R and T). It is not known whether there is a causal relationship between this change in expression and the sudden expansion and branching of the pancreas epithelium occurring from e11.5.

At e12.5, the gut has rotated and the two pancreatic rudiments have fused to become one interconnected organ (Fig. 2O). This process is not well understood, but when it is disrupted as in hedgehog mutants, malformations such as annular pancreas can develop (102, 103). Upon fusion, the connection of the dorsal pancreas to the duodenum becomes noticeably smaller than the corresponding ventral connection that eventually will form the main duct.

B. The peptide hormones
The endocrine part of the pancreas consists of five different cell types, each characterized by distinctive expression of one of the specific peptide hormones: glucagon (Gcg) in the -cells, insulin (Ins) in the ß-cells, somatostatin (Sst) in the -cells, pancreatic polypeptide (Ppy) in the PP-cells, or ghrelin (Ghrl) in the -cells (61, 104, 105, 106). The onset of expression of the peptide hormones in mice has been determined by RT-PCR to be at eight somites for somatostatin (but not specifically within the pancreas domain), at 20 somites for insulin and glucagon, and at 30 somites for Ppy (107). Moreover, the single-cell RT-PCR analysis of e10.5 dorsal pancreatic cells reveals the presence of glucagon, insulin, somatostatin, and Ppy transcripts in various combinations of coexpression (90). However, the immunodetection of the peptides and the issue of coexpression in the early endocrine cells have been debated over the years.

The earliest time point for immunodetection has been reported to be at e9.5 for glucagon and insulin (108), at e13.5 for somatostatin, and at e10.5 for ghrelin and Ppy (105, 109). Others have reported that Ppy cannot be detected until after birth and that embryonic immunodetection of Ppy is due to cross-reactivity of the antibodies to the related neuropeptide Y (Npy) and peptide YY (Pyy) peptides (108, 110).

We find that all the islet peptides except Ppy can be detected in the dorsal pancreas as early as e9.5, with glucagon being the most abundant (Fig. 3, A, E, I, and M). Cells expressing both insulin and glucagon can be found, but it is also evident that cells only expressing one of the two hormones are present (Fig. 3A). Previously, it has been suggested that the double-positive cells were precursors for the mature - and ß-cells (111, 108). However, expression analysis of endocrine markers suggests that - and ß-cells develop independently (112) and also that lineage tracing and cell ablation studies have demonstrated that the double hormone-expressing cells are not required for the subsequent formation of - and ß-cells (113, 114). The fate of the double hormone-positive cells remains unknown. There are also significant amounts of ghrelin-expressing cells present already at e9.5 (Fig. 3E), and the numbers relative to glucagon-expressing cells seem to peak at e11.5 (Fig. 3, E–H). At these stages, many of the ghrelin-expressing cells do not coexpress glucagon (Fig. 3. E–H). This observation does not fully correlate with a previous study showing that ghrelin is predominantly coexpressed with glucagon during embryonic pancreas development (105), which may possibly be due to differences between mouse strains. The lineage relationship of the mature -cells to the other endocrine cell types has not been addressed.

View larger version (49K): [in this window] [in a new window]   FIG. 3. Expression of pancreatic endocrine peptide hormones and an exocrine marker. Collapsed image stacks of wild-type embryos stained for the expression of Gcg and Pdx1 in whole embryos (e9.5 and e10.5) or in dissected pancreata (e11.5 and e12.5). At e12.5, only the dorsal pancreata are shown. The specimens were costained for the expression of insulin (Ins; A–D), ghrelin (Ghrl; E–H), somatostatin (Sst; I–L), pancreatic polypeptide (Ppy; M–P), peptide YY (Pyy; Q–T), and Cpa (U–Y). A, At e9.5, both Ins and Gcg can be detected in the dorsal pancreas. Gcg-expressing cells are more abundant, and a subset of the Gcg-positive cells also expresses Ins. Most of the Ins-positive cells express Gcg, but single positive cells can be observed. A–D, Common for both Ins- and Gcg-expressing cells is that they seem to cluster and accumulate at the tip of the growing pancreas. D, Although single positive Ins cells can be detected, these do not express high levels of Pdx1, suggesting that they are not mature ß-cells. C, G, K, O, and S, Gcg-expressing cells appear in the ventral pancreas at e11.5. E–G, Ghrl-positive cells are rare at e9.5, but over the next couple of days they increase in number, and at e11.5 they represent about half of the endocrine population. H, At e12.5, the number of Ghrl-positive cells decrease. Throughout, a fraction of the Ghrl-positive cells is also positive for Gcg, but many only express Ghrl. As also observed for Ins and Gcg, Ghrl-positive cells seem to cluster at the tip of the dorsal pancreas. I, A few Sst-expressing cells can first be detected at e9.5 (arrows). These cells express low levels of Sst and are best seen on the optical sections (inset). J–L, Over the next days the Sst-positive cells can be detected reliably, but always in very low numbers where some coexpress Gcg while others do not. M–P, Ppy behaves slightly different from the other hormones and cannot be detected until e11.5, when it suddenly appears in both the dorsal and ventral pancreas rudiments. O and P, In contrast to the observed expression patterns for Ins, Gcg, and Ghrl, Ppy-expressing cells lie scattered within the central (Continued) epithelium and do not cluster at the tip. O, Also, Ppy-expressing cells are the most predominant endocrine cell type in the ventral pancreas at e11.5. Coexpression with Gcg can be detected, but not always (O and P; also see supplemental data). Q–T, Pyy is expressed extensively from e9.5 to e12.5. Almost all Gcg-positive cells coexpress Pyy, but a substantial number of cells in the ventral pancreas (S) and in the central part of the dorsal pancreas (T) are single positive for Pyy. U and V, Cpa can first be detected in both the dorsal and ventral buds at e10.5. However, the expression is low and is best observed on the optical sections (V, inset; also see supplemental data). X and Y, At e11.5 and e12.5, Cpa is not coexpressed with Gcg but is otherwise expressed at high levels in cells adjacent to the mesenchyme. In all panels, anterior is to the right, posterior is to the left, dorsal is above, and ventral is below.

C. The exocrine pancreas
The acinar cells of the pancreas produce carboxypeptidases, amylase, chymotrypsin, trypsin, ribonucleases, and lipases, which are digestive enzymes responsible for the hydrolytic degradation of carbohydrates, nucleic acids, proteins, and fatty acids in the digestive tract (6). Carboxypeptidase A (Cpa) and elastase are considered to be early exocrine markers, whereas amylase is a late marker, and the sheer rise in the pancreatic content of exocrine products from e14.5 in rats was the basis for the definition of the secondary transition (6, 93). The first lineage ablation study addressing pancreas development was transgenic mice with elastase promoter-driven diphtheria toxin A expression. The resulting phenotype demonstrated arrested pancreas development around e14.5 when expression of diphtheria toxin A was activated, suggesting that proliferation of duct and endocrine cells depends on differentiation of the acinar cells (117).

Cpa can be detected by RT-PCR and immunohistochemistry from e10.5 (90, 107) (Fig. 3V, inset). At e11.5, it is expressed at high levels in both the dorsal and ventral pancreas (Fig. 3X). In contrast to the expression patterns of the endocrine-specific peptides, there is no delay in the ventral bud compared with the dorsal and it seems to be a significant part of the undifferentiated pancreas epithelium that expresses Cpa. Thus, Cpa is not a specific marker of exocrine differentiation at this time point, although it would require lineage tracing to conclusively demonstrate that Cpa is also expressed in endocrine precursor cells as indicated by coexpression with Neurog3 and other endocrine markers as well as the endocrine hormones in the single-cell RT-PCR analysis (90) (see Note Added in Proof). At e12.5, the expression of Cpa has segregated to the forming acini at the periphery (Fig. 3Y). Amylase is expressed from e13.5 as detected by RT-PCR and immunohistochemistry (107).

	IV. Transcription Factor Signature of the Multipotent Pancreatic Progenitor Cells

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Pancreas formation is dependent on several successive requirements to be fulfilled. First, prepatterning of the endoderm and selective suppression of sonic hedgehog expression in the pancreatic domain is prerequisite (55, 60, 118). Also, the pancreatic endoderm must receive appropriate signals from neighboring tissues (38, 39, 63, 67, 69), and finally, the epithelial expression of the key transcription factors Pdx1, Ptf1a, and Hlxb9 is critical (43, 45, 85, 86, 87).

A. Pdx1
Regarding pancreas development, the first important transcription factor to be identified was the pancreatic and duodenal homeobox 1 (Pdx1) which was originally found to be a critical transcriptional activator of insulin and somatostatin in adult islet cells (44, 119, 120, 121, 122). The specification of endoderm to a pancreatic fate does not rely on Pdx1 function, but it is necessary for development beyond initial bud formation. Thus, Pdx1 deficiency in mouse and human results in arrested growth of the pancreatic primordia at a very early stage, leading to complete pancreas agenesis at birth (85, 87, 123, 124). Also, a few early glucagon- and insulin-positive cells can develop independently of Pdx1 (42). The pancreatic mesenchyme in Pdx1-deficient mice develops normally, suggesting that in the absence of Pdx1 the epithelium is unable to respond to the mesenchymal signals that normally promote growth and differentiation (42). Furthermore, correct levels of Pdx1 expression are important for proper pancreas formation (125).

Only a few forms of diabetes can be attributed to monogenetic variation. These are known as maturity onset diabetes of the young (MODY), and Pdx1 has been identified as the MODY4 gene (126). Also, conditional inactivation of Pdx1 in adult mouse ß-cells results in diabetes (127). This demonstrates a requirement for Pdx1 not only during pancreas development, but also for proper ß-cell function and maintenance.

Strong expression of Pdx1 can be observed as early as e8.5 (43, 128) in two ventrolateral domains before closure of the foregut endoderm (Fig. 2, A and B). Slightly later at e8.75, a dorsal Pdx1 expression domain appears at the level of somite pair number 4–7 just after passage of the anterior intestinal portal and the closure of the foregut endoderm (Fig. 2, C and D). Pdx1 is specific for the foregut epithelium and is not found to be expressed anywhere else in the embryo proper. Until e9.5, Pdx1 expression is restricted to the pancreatic domains of the foregut (Fig. 2, E–J), and thereafter it is also found in the epithelia of the duodenum, the bile duct, and the posterior part of the stomach (43, 87, 120, 128) (Fig. 2, K, M, and O). At e13.5, a few cells with significantly higher Pdx1 expression levels can be found in the central epithelium, and this becomes more prominent at e14.5 (Fig. 2, Q–T). These constitute the forming ß-cells (112).

B. Hlxb9
Another critical homeobox gene product, Hlxb9 (Hb9), was identified in the pancreas (129), and analysis of Hlxb9-deficient mice reveals a complete agenesis of the dorsal pancreas whereas the ventral pancreas has no defects in the development of the epithelium and displays only a late phenotype resulting in immature ß-cells and disorganized islet structure (43, 45). Hlxb9 is expressed throughout the dorsal endoderm before Pdx1 in the presumptive pancreatic endoderm (Fig. 4A1 and inset). From e9.0 to e9.5, it becomes gradually restricted to the dorsal pancreatic epithelium where it is coexpressed with Pdx1 (Fig. 4, A1–A3). At e9.5, it starts to appear in the ventral pancreas (Fig. 4A3, inset). The expression level of Hlxb9 peaks at e10.5 in the epithelia of both dorsal and ventral pancreas, and additionally, it is seen in the anterior dorsal stomach epithelium and posteriorly in the dorsal gut epithelium with boundaries to the Pdx1 expression domain (Fig. 4A4). At e11.5 and more pronounced at e12.5, Hlxb9 expression declines in the periphery but remains in some cells in the central pancreatic epithelium, and at later stages, it becomes restricted to the ß-cells (43, 130). Persistent Hlxb9 expression under the control of the Pdx1 promoter leads to impaired pancreas development (130). Thus, temporal regulation of Hlxb9 expression is essential for proper pancreas development. Hlxb9 is also expressed in differentiated motor neurons of the spinal cord (131, 132) and in the notochord (45) (Fig. 4A1) and defective notochord function could be an alternative explanation of the selective dorsal pancreas agenesis observed in the knockout.

C. Ptf1a
The pancreas-specific transcription factor 1a subunit Ptf1a (p48) is an important bHLH factor, functioning as part of a trimeric protein complex in the regulation of exocrine gene transcription (89, 133). It is coexpressed with Pdx1 in both dorsal and ventral pancreas epithelia from e9.0 to e9.5 (Fig. 4, B1–B3). At e10.5, Ptf1a is restricted to the epithelium of the pancreatic primordia with sharp boundaries to the duodenum (Fig. 4B4). Later at e12.5, Ptf1a expression segregates to the growing tips of the branching epithelium (Fig. 4B6) to eventually end up in the acinar cells.

Ptf1a deficiency results in pancreas agenesis, but like Pdx1, Ptf1a is not necessary for the initial pancreas formation (86, 88). There is a complete absence of exocrine development, but early endocrine cells can still be found (86). Lineage tracing has shown that cells normally contributing to the ventral pancreas adopt an intestinal fate in the absence of Ptf1a (88). The initiation of Ptf1a expression appears to be selectively induced by a signal from endothelial cells because Flk1 mutants that lack all endothelial cells (134) fail to induce Ptf1a, a phenotype that can be rescued in coculture explants with wild-type aorta (63). However, this might be an effect that is mediated through the dorsal pancreatic mesenchyme because the endothelium of the dorsal aorta seems to support the survival of the mesenchymal cells (135, 136) from which FGF10 signaling in turn is necessary to sustain Ptf1a expression in the dorsal pancreatic bud (136). Later, Notch dependent repression of acinar cell differentiation ensures continuant growth of the pancreatic epithelium, and it has been suggested that this is mediated via direct interactions between Ptf1a and Hes1, resulting in inhibition of the trimeric Ptf1 functional activity (76, 137). Alternatively, it may be due to a competition during development between the intracellular domain of Notch and Ptf1a for binding to the recombination signal binding protein Rbpj (RBP-J), which normally mediates the transcriptional effects of Notch signaling (138). In mature acinar cells, the Ptf1 complex contains the Rpbj paralogue, Rbpjl (RBP-L), which is responsible for the Notch signaling independent high transcriptional activity of the acinar-specific genes (138).

D. Nkx6-1 and Nkx2-2
The homeodomain transcription factors Nkx6-1 and Nkx2-2 are other early markers of the pancreatic epithelium (139, 140, 141). In mature islets, Nkx6-1 expression is restricted to the ß-cells (142) where it has been suggested to act as a transcriptional repressor of glucagon promoter activity (143, 144), whereas Nkx2-2 marks the -, ß-, and PP-cells (145). During embryonic development, both are also expressed in the ventral part of the neural tube (146, 147), and Nkx6-1 is additionally found in the mesenchyme surrounding the esophagus and anterior stomach epithelium (33) (Fig. 2, E–S).

The earliest detection of endodermal Nkx6-1 expression is at e8.75 in the prospective ventral pancreas domain where a few cells appear transiently within the Pdx1 domain (Fig. 2D, inset). At e9.0, the first few cells of the dorsal Pdx1 domain have initiated Nkx6-1 expression, and at this time point Nkx6-1 immunoreactivity can no longer be detected in the ventral domain (Fig. 2, E and F). Ventral Nkx6-1 reappears at e10.5, where the expression pattern of Nkx6-1 resembles that of Ptf1a by being highly restricted to the dorsal and ventral pancreatic epithelia and with sharp boundaries to the bile duct and the duodenal epithelium (Fig. 2, K and L). At e11.5, it starts to become restricted to the central epithelium (Fig. 2, N, P, R, and T), reciprocally to Ptf1a which segregates to the epithelium of the growing tips and the developing exocrine tissue. At e13.5 and e14.5, some of the cells in the central epithelium demonstrate significantly higher Nkx6-1 expression levels than in general.

Pancreatic expression of Nkx2-2 is initiated together with dorsal Pdx1 at e8.75 (Fig. 4D1, inset) and ventral Nkx2-2 expression appears at e9.5 (Fig. 4D3). Most of the pancreatic epithelial cells are positive for Nkx2-2 until e11.5, just as this is observed for Nkx6-1 and Ptf1a, but positive cells vary in intensity with strong and weak nuclear staining intermingled (Fig. 4, D1–D4; see also supplemental data). The high-level Nkx2-2-expressing cells represent endocrine progenitor cells, and the lower level Nkx2-2 expression follows the Nkx6-1 expression pattern and disappears from the growing tips of the epithelium at e12.5 (Fig. 4D6).

Nkx6-1 deficiency results in compromised ß-cell development (140), but apparently it does not affect early pancreas development because these mice do not show any early phenotype and the pancreas morphology appears intact (140). The closely related Nkx6-2 and Nkx6-3 are also expressed in the developing foregut endoderm (33, 141), and there is evidence of redundancy because Nkx6-2 can compensate partly for the lack of Nkx6-1 (148, 149). Although the Nkx6-1/6-2 double mutants display a stronger phenotype than either of the single mutants, the early developmental events of pancreas formation are intact (149).

Nkx2-2 deficiency results in loss of the ß-cells and reduced numbers of - and PP-cells, whereas the number of ghrelin-positive cells increase, suggesting a replacement of the ß-cells with -cells in the absence of Nkx2-2. There is no impairment of the -cells, and there is no early phenotype because both Pdx1 and Nkx6-1 expression is maintained in the epithelium until e14.5 (106, 145, 150). Nkx2-2/Nkx6-1 double mutants have been generated and analyzed, and they demonstrated a phenotype identical to the Nkx2-2 single mutants, excluding the possibility of redundant functions between the two factors in the early pancreas epithelium (140).

	V. Pancreatic Endocrine Progenitor Cell Determination

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The three principal pancreatic tissue types that serve very different purposes originate from a common endodermal progenitor cell. It was debated for many years whether the endocrine cells could be of neural crest origin because neuronal cells and endocrine cells express several common markers. However, cell tracing experiments with chick-quail chimeras has demonstrated that migrating neural crest cells never give rise to endocrine cell types in the pancreas but differentiate into parasympathetic ganglia in the mesenchyme (see Refs. 151 and 152 ; for review, see Ref. 153). Since then, molecular lineage tracing has shown that all pancreatic lineages are derived from cells expressing Pdx1 and Ptf1a (85, 88, 154). At e10.5, cells expressing these factors also express Hlxb9, Nkx6-1, and Nkx2-2 as described above. Throughout pancreas development, the commitment of cells to a particular lineage represents a fine balance between proliferation and differentiation. Notch receptor signaling in the pancreatic epithelium inhibits both exocrine and endocrine differentiation, and inhibition of Notch signaling results in accelerated differentiation at the expense of organ growth (73, 75, 76, 77, 78, 80). The presumptive Notch target gene Hes1 may act at least partly by preventing p57-mediated cell cycle exit required for differentiation during early stages of pancreas formation (155).

A. Neurog3
The bHLH transcription factor Neurog3 is expressed in pancreatic endocrine progenitor cells and is required for endocrine differentiation, as demonstrated by the complete lack of endocrine cells in Neurog3-deficient mice (112, 154, 156, 157). Neurog3 is also expressed in enteroendocrine progenitor cells of the gut and in neuronal progenitor cells in the neural tube (158, 159).

Lineage tracing has shown that transiently expressing Neurog3 cells during pancreas development indeed are endocrine progenitors and give rise to -, ß-, -, and PP-cells (154). Furthermore, ectopic expression of Neurog3 or its downstream target Neurod1 is sufficient to induce differentiation of endocrine cells in the mouse pancreas, chicken endoderm, and in primary human pancreatic duct cells and duct cell lines (47, 73, 157, 160).

At stages between e8.5 and e8.75, a few Neurog3-positive cells can be detected among the thin dorsal stretch of newly emerged Pdx1 cells (data not shown). At e9.0, 50–60 Neurog3-positive cells can be detected within the Pdx1 domain in the dorsal pancreatic epithelium, but a few of them are often found outside the Pdx1 domain, suggesting that some of the early endocrine cells differentiate independently of Pdx1 (Fig. 4C1), as also demonstrated by the presence of endocrine cells in Pdx1 null mice (42). Ventral Neurog3 expression first appears at e9.5 (Fig. 4C3) and from e11.5, Neurog3 expression is primarily located in the central pancreas epithelium (Fig. 4, C5 and C6). Pancreatic Neurog3 expression is restricted to embryonic life and disappears around the time of birth (156).

B. Nkx2-2
The expression pattern of Nkx2-2 with diverse expression intensities in the developing pancreas epithelium is rather unusual and has to be divided into two subpopulations where the high-level Nkx2-2-expressing cells characterize endocrine progenitor cells and the lower level Nkx2-2 expression is characteristic of the undifferentiated epithelium (Fig. 4, D1–D6 and supplemental data). Until e9.5, scattered Nkx2-2-positive cells can be observed in the duodenum outside the pancreas areas (Fig. 4, D1–D3), and a subset of the high-level Nkx2-2-expressing endocrine progenitor cells is found not to coexpress Pdx1 (Fig. 4, D1–D3; also see supplemental data). This correlates with the data set from the single cell transcript analysis of e10.5 pancreas presenting examples of Neurog3- and Nkx2-2-positive cells without Pdx1 (90).

In contrast to the early epithelial expression of Nkx6-1, which is unaffected in Nkx2-2-deficient mice, ß-cell-specific Nkx6-1 expression is lost in the endocrine cells of Nkx2-2 mutants, demonstrating that Nkx6-1 acts downstream of Nkx2-2 in ß-cell differentiation (145). In neuronal development, Nkx2-2 has been shown to act as a repressor through the recruitment of Tle corepressors (161). In particular, Tle3 (Grg3) is expressed in the pancreas and has been shown to physically interact with Nkx2-2 in vitro, and an Nkx2-2-dominant-repressor transgene can fully restore the -cell mass but is only partly able to rescue the Nkx2-2 mutant ß-cell phenotype (162). The recovered insulin-positive cell numbers are low compared with wild type, and they express Nkx6-1 but not the terminal ß-cell markers Mafa and Slc2a2 (Glut2), indicating that Nkx2-2 might work through alternative cofactor interactions or have additional activator functions (162).

C. Pax6
The paired domain transcription factor Pax6 has been reported to mark all pancreatic endocrine cells from e9.0, and Pax6 deficiency compromises all the endocrine cell types, although primarily the -cells (163, 164, 165). At this stage, we do not observe Pax6-positive cells in the pancreatic epithelium (Fig. 4E1); however, slightly later at e9.25 (16 somites) we observe robust expression of Pax6 (Fig. 4E2) and at e10.5, the first few Pax6 cells appear in the ventral pancreas (Fig. 4E4). At e11.5, the Pax6 cells start to cluster (Fig. 4E5), and this becomes more evident at e12.5 (Fig. 4E6). Pax6 continues to be expressed in the mature endocrine cells where it functions as a transcriptional activator of several of the endocrine hormone genes (164, 166, 167). Additionally, Pax6 is expressed in the developing brain, the neural tube, and the eyes (165).

D. Pou3f4
The POU-homeodomain factor Pou3f4 (Brn4) has been reported to be a pancreatic -cell-specific transcription factor (163, 168). At e9.0 and e9.25, strong nuclear Pou3f4 expression is found in a scattered pattern within the dorsal Pdx1 domain but also occasionally outside (Fig. 4, F1 and F2). Apparently, the onset of Pou3f4 expression is before the initiation of Pax6. Closer inspection reveals that almost all the Pou3f4-positive cells at these early stages are devoid of Pdx1, but from e9.5 the segregation of Pou3f4- and Pdx1-expressing cells becomes less prominent (Fig. 4, F3 and F4). At e10.5, Pou3f4 expression is detected in the ventral pancreas and again Pou3f4-positive cells can be found outside the pancreas epithelia (Fig. 4F4). At e11.5, some of the dorsal Pou3f4 cells emerge in clusters, but there are also many scattered Pou3f4 cells in the duodenum and the bile duct (Fig. 4F5). At e12.5, the clustering of the Pou3f4 cells is evident, just as it is observed for Pax6 (Fig. 4F6).

Pou3f4 functions as a transcriptional activator of glucagon gene expression (168, 169), but is apparently not essential during pancreas development because Pou3f4 mutant mice display normal pancreas morphology and cytodifferentiation. Also, -cell secretion of glucagon is normally regulated (163), and it is possible that a similar factor serves redundant functions but this has not been addressed.

	VI. Pancreatic Endocrine Cell Fate Determination

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The cellular switch toward an endocrine program is designated by Neurog3 expression (112, 156). Thus, Pdx1 promoter-controlled ectopic Neurog3 expression in early mouse pancreas progenitor cells results in substantial induction of endocrine differentiation but exclusively to -cells (73, 157). These results suggest that progenitor cells at early time points have a limited competence for endocrine differentiation. However, alternative interpretations regarding Neurog3 expression levels or duration of Neurog3 activity present other plausible explanations of the observed phenotype given the experimental conditions. Recent data elegantly demonstrate that, indeed, the competence of the progenitor cells changes over time (170). Conditional activation of Pdx1 promoter-driven Neurog3 expression in a Neurog3 null background shows that activation of Neurog3 at the onset of Pdx1 expression (e8.75) results in the exclusive formation of -cells. The competence to form ß-cells and PP-cells is acquired after e10.5, and -cells only appear when Neurog3 activity is induced from e14.5. The relative competence to form -cells becomes strongly reduced after e14.5, but lineage tracing of all endocrine cells formed after e14.5 has revealed that approximately 12% of these acquire an -cell fate (170). Hence, a significant contribution to the final number of -cells seems to develop between e14.5 and e16.5 because the absolute number of endocrine progenitor cells in mouse pancreas peaks in this time window. Ectopic induction of Neurog3 at e10.5 and subsequent isolation of the epithelium results exclusively in -cell differentiation when reassociated with e14.5 wild-type mesenchyme, suggesting that the competence of the endocrine progenitors is autonomous to the pancreas epithelium and independent of signals from the mesenchyme (170). This is in contrast to previous findings indicating that the mesenchyme controls the timing of ß-cell differentiation in cultured pancreas explants (171).

Because Neurog3 is able to induce all the endocrine subtypes, additional factors must be involved in specific cell fate assignment. Pax4 and Arx are two recently identified factors with such properties. Loss of either of these genes or the simultaneous loss of both genes in the mouse does not affect the total number of endocrine cells but rather changes the relative distribution of endocrine subtypes (115, 116, 172). Pax4 transcripts can be detected in the mouse pancreas as early as e9.5 by mRNA in situ hybridization (115). Pax4 has been inactivated by an in-frame fusion of the ß-galactosidase gene to the N terminus of the gene by homologous recombination in the mouse (115), and LacZ activity or immunodetection of ß-galactosidase has been used to investigate the expression pattern of Pax4 in mice heterozygous for this allele. From e11.5, Pax4 is expressed in scattered cells within the pancreas epithelium, and the number of Pax4 cells peaks during the secondary transition where it is found to be coexpressed with a number of endocrine markers including Neurog3 and Isl1 (150). At e18.5 and in neonates, most Pax4-expressing cells coexpress insulin, whereas only a few express glucagon (115, 150). During embryogenesis, Pax4-deficient mice display a selective loss of ß-cells and -cells with a concomitant proportional increase in -cell numbers, suggesting that cells that normally acquire a ß- or -cell fate adopt an -cell fate in the absence of Pax4 (115). The use of antibodies against ß-galactosidase to detect Pax4 expression indicates that Pax4 is down-regulated at the end of gestation and cannot be detected in adult mouse pancreas (150). However, this is controversial because others report that Pax4 is expressed in adult rat and human islets using RT-PCR to detect the mRNA (173). Whether this discrepancy represents species variation or differences in assay sensitivity or reflects that only a subpopulation of cells in the pancreas express Pax4 waits to be resolved. Loss of Pax4 seems to block ß-cell differentiation at an early stage, as indicated by the absence of Pdx1, Hlxb9, and insulin expression from ß-cell precursors (150). In contrast, Nkx2.2 is unaffected in Pax4 null mice and because Nkx2.2 deficiency results in a similar loss of Pdx1 and Hlxb9 expression from ß-cell precursors without affecting the expression of Pax4, it has been suggested that Pax4 and Nkx2.2 regulate parallel pathways in the initiation of the ß-cell differentiation program (150). Loss of either Pax4 or Nkx2.2 results in an increase in the number of ghrelin-positive cells, but in the case of the Pax4 mutants, most of these cells coexpress glucagon and therefore most likely represent transdifferentiated -cells that would normally have become ß-cells. The number of -cells expressing only ghrelin is unchanged in Pax4-deficient mice compared with normal mice (105, 106). Although inactivation of Pax4 only affects the ß- and -cell lineages, it has recently been shown that Pax4 is expressed in progenitors of all endocrine cell types, suggesting that Pax4 expression is activated early in the differentiation cascade and that it is not Pax4 as such that determines the cell fate (80). It has been shown in vitro that Neurog3 is capable of activating the Pax4 promoter (174).

Arx transcripts can first be detected at e9.5 in the mouse pancreas by mRNA in situ hybridization. From e12.5, it becomes restricted to the forming islets, and the expression is completely lost in Neurog3 null mice (116). Arx deficiency results in complete absence of -cells at e15.5 as well as in neonates that die 2 d after birth as a result of hypoglycemia. However, the early glucagon-expressing cells form in normal numbers at embryonic stages before e15.5 (116), suggesting that either the -cells born before e15.5 disappear during normal development or that Arx function is required for the survival or maintenance of these -cells. Arx-deficient mice display an increase in the number of insulin-producing cells from e15.5 and, similarly, an increase in -cell numbers from e18.5 (116). This is the opposite phenotype to the Pax4-deficient mice, and it has been demonstrated in vitro that Arx and Pax4 can mutually cross-repress each others’ promoters, directly suggesting a mechanism where Arx and Pax4 compete in the endocrine precursors (172). Loss of Arx does not affect the number of -cells (105).

Analysis of the endocrine pancreas in Pax4/Arx double homozygous mice shows that the simultaneous loss of both genes results in an early onset loss of both - and ß-cells as would be expected from the single mutant phenotypes (172). The - and ß-cells are replaced with -cells, suggesting that Pax4 acts sequentially to first promote the formation of a ß/-cell precursor pool and subsequently is required specifically for the development of ß-cells from these precursors. Ppy-expressing cell numbers are normal in these embryos, but after birth, massive induction of Ppy expression occurs in the supernumerary -cells as a response to feeding (172).

All these data demonstrate that the endocrine differentiation process is highly plastic and raise the question whether differentiated cells also have the potential to transdifferentiate. This issue has recently been addressed by conditional activation of Arx in either pancreatic progenitor cells (crossed with pPdx1-Cre), in endocrine precursors (crossed with pPax6-Cre), or in differentiated ß-cells (pIns-Cre or an inducible pPdx1-Cre) (175). The data demonstrate that Arx is not only required but also sufficient to specify -cell fate in the embryonic pancreas. Thus, activation of Arx expression in the pancreatic progenitor cells and in the endocrine precursors results in a massive increase in -cell numbers and, somewhat surprising, also in the number of PP-cells with a concomitant decrease in ß- and -cell numbers. One week after induction of Arx expression in mature ß-cells, insulin immunoreactivity disappears in the islets with a concomitant increase in the number of glucagon and Ppy-positive cells, and the mice eventually die from hyperglycemia. Lineage tracing showed that the increase in - and PP-cells is derived from cells that have previously expressed insulin and Pdx1, indicating a greater plasticity of the endocrine cells than previously thought (175).

Other factors such as Mafa and Mafb of the v-maf musculoaponeurotic fibrosarcoma oncogene family and the forkhead gene Foxa2 may also play roles in endocrine cell fate determination. At early embryonic stages, Mafb is expressed in the glucagon and the insulin-positive cells, but around the secondary transition, it becomes down-regulated in the insulin-positive cells where Mafa appears. Mafb becomes restricted to the -cells where it controls glucagon gene expression (176, 177, 178). Mafa is an activator of insulin gene expression and appears to be specific for developing and adult ß-cells (176, 179, 180, 181). Mouse embryos lacking functional Foxa2 die around e10 (182, 183), and the morphology of the embryos does not permit the pancreas to be analyzed. However, conditional inactivation of Foxa2 in the endoderm using a Foxa3 transgene to drive Cre recombinase recently revealed that early pancreatic markers such as Pdx1 are unaffected by the absence of Foxa2 (184). At later stages, the expression of glucagon is severely reduced, but the mRNA levels of the -cell transcription factors Arx and Pou3f4 (Brn4) are unaffected (184). Also, cells expressing the prohormone convertase Pcsk2 (PC-2) but not insulin or somatostatin are found at the periphery of the islets where the -cells would normally reside (184), suggesting a role for Foxa2 in -cell maturation.

	VII. Summary and Future Aspects

Top Abstract I. Introduction II. From Endoderm to... III. Pancreatic Endocrine and... IV. Transcription Factor... V. Pancreatic Endocrine... VI. Pancreatic Endocrine Cell... VII. Summary and Future... Note Added in Proof References

 
The turning of the mouse embryo at e8.75 is a key point in the developmental process and is a very dynamic process that rearranges the organization of the different tissues surrounding the pancreatic endoderm. Structures that are known to provide pancreas-promoting signals like the notochord and the blood vessels can be visualized using specific markers, and the positioning relative to the developing pancreas can easily be examined in 3D as exemplified here. These dynamic processes of tissue reorganization happen during the primary transition period between e8.5 and e10.5, first described by Rutter and co-workers (6, 93). The collective expression patterns of the transcription factors that mark the early pancreatic epithelium are interesting, and the combinatorial code of Pdx1, Hlxb9, Ptf1a, Nkx6-1, and Nkx2-2 expression is unique for the pancreatic epithelium in this transition phase (Fig. 5). It is, however, rather unclear how these factors function at the molecular level. Pdx1 is the only pancreatic transcription factor known that is strictly confined to the endoderm without being expressed in the neural tube or any other tissue. Nevertheless, Pdx1 is not strictly pancreas-specific because it is also expressed in visceral endoderm before gut closure and in the epithelia of the posterior stomach, the duodenum, and the bile duct from e10.5. Interestingly, recent work reports that Nkx6-2, a close relative to Nkx6-1, shows a similar expression pattern (33, 141). The dynamic expression of Hlxb9 is also noteworthy. It is clearly indispensable for specification of the dorsal pancreas (43, 45), but it cannot be excluded that this could be due to a notochord malfunction rather than a direct effect within the dorsal pancreas epithelium. Outside the pancreas area, Hlxb9 expression disappears from the Pdx1 domain but remains in more anterior and posterior dorsal endoderm (Fig. 4A4). Ptf1a and Nkx6-1 are coexpressed and much more restricted to the pancreatic epithelia in the early developmental phase from e9.0 to e11.5. Thereafter, they segregate and become mutually exclusive in the epithelium, where Ptf1a becomes confined to the peripheral growing tips and Nkx6-1 to the central part of the epithelium (Fig. 5). In both the dorsal and the ventral pancreas buds, the onset of Hlxb9 and Nkx2-2 expression precedes Nkx6-1 but at later stages until e12.5 both follow the expression pattern of Nkx6-1 (Fig. 5).

View larger version (26K): [in this window] [in a new window]   FIG. 5. Correlations between morphogenetic events and pancreatic gene expression patterns. A schematized diagram illustrating dorsal pancreas development from e8.5 to e12.5 in the left column (only dorsal pancreas and not ventral pancreas is illustrated) with color-coded expression profiles of the key transcription factors listed to the right. A, Hlxb9 (red) is expressed in dorsal endoderm before initiation of dorsal pancreas organogenesis. Onecut1 is also known to be expressed in the endoderm before dorsal Pdx1 expression. B, The first dorsal Pdx1-positive cells (green) appear at the level of somite pairs 4 to 7 simultaneously with the turning of the embryo between e8.5 and e8.75 but before actual thickening of the endoderm. Then the dorsal bulge starts to appear. C, Nkx2-2 (green) becomes coexpressed with Pdx1 (green) as early as e8.75, and already at e9.0 many cells turn on Neurog3 expression and enter the endocrine pathway. D, Hlxb9 (red), Pdx1 (green), low-level Nkx2-2 (here in red), and also Nkx6-1 (red) and Ptf1a (blue) are all coexpressed in the dorsal bud from e9.25 to e10.5, but Pdx1 (green) ceases to be pancreas restricted at e10.5 and appears in the duodenal epithelium. E, At e11.5 and more pronounced e12.5, the entire pancreatic epithelium is still Pdx1 (green) positive, but the other transcription factors Nkx6-1, low-level Nkx2-2, and Hlxb9 (all in red; Hlxb9 is within parentheses because it is generally down-regulated in the epithelium except in some scattered cells) segregate to the central epithelium, whereas Ptf1a together with Cpa (both in blue) are specifically found in the periphery. This segregation of expression patterns accompanies a morphological change from a uniform epithelial structure to a branching structure where the Ptf1a- and Cpa-positive branching tips have become committed to an exocrine pathway and will form acinar cells. Hes1 inhibits the exocrine differentiation process. At all time points from e9.0, cells continuously enter the endocrine pathway by initiation of Neurog3 expression and succeeding endocrine lineage markers like high-level Nkx2-2, Pou3f4, Pax4, Arx, Neurod1, Isl1, and Pax6. At e11.5 and e12.5, this happens only from the central part of the epithelium. Hes1 is also known to inhibit the endocrine differentiation process. It is unknown which cell population gives rise to the mature duct cells (marked by dotted lines and question marks). Factors that have not been included as stainings in the previous figures are shown in italics.

Another key point in pancreas development seems to be at e11.5, where there clearly is a switch in the growth process from a uniformly Pdx1, Ptf1a, Hlxb9, Nkx6-1, and Nkx2-2 patterned pancreatic epithelium to a rapidly expanding Pdx1-positive structure with reciprocal compartments of a Hlxb9, Nkx6-1, and Nkx2-2-expressing central epithelium where endocrine cells arise and Ptf1a-expressing branching tips that are committed toward an exocrine fate (Fig. 5). The exocrine marker Cpa also becomes confined to the forming acini, and forced expression of Neurog3 in these cells is unable to activate an endocrine program (170), suggesting that an irreversible change toward a phenotype with restricted developmental potential occurs. Conversely, transgenic mice with Pdx1 promoter-controlled expression of Hlxb9 show perturbed pancreas growth from e11.5 (130), presumably as a result of ectopic Hlxb9 expression in the peripheral growing tips.

All the endocrine cell type-specific peptide hormones as well as the exocrine marker Cpa are detectable several days before the secondary transition beginning at e13.5, and it is evident that cytodifferentiation in the sense of initiation of cell-specific gene expression occurs already in the earliest phases of pancreas development. This points toward a model of a more extended differentiation process that commences earlier than previously thought and without a defined intermediate period of a protodifferentiated state. For the endocrine lineage, it seems to be a consecutive and perhaps stepwise process where the expression of a certain peptide hormone is not necessarily a marker of terminal differentiation. The whole organ analysis reveals that significant numbers of cells express glucagon, insulin, and ghrelin already from e9.5, with glucagon being the most abundant. It also shows that only part of the cells expressing either insulin or ghrelin coexpress glucagon. The fate of the insulin and glucagon double-positive cells is unclear, and that is also true for the ghrelin cells in general, which are present at relatively high numbers at e10.5 and e11.5. Lineage tracing of these ghrelin-expressing cells could potentially provide important information about their properties and fate. The appearance of significant amounts of Ppy-expressing cells at e11.5 agrees well with previous expression data (107, 109), and the lineage-tracing study suggesting that Ppy expression is an intermediate step in the formation of ß- and -cells (114). The order of appearance of the hormones matches with recent data showing that the pancreatic epithelium acquires a temporally graded competence to generate cells expressing first glucagon from e8.5, then insulin and Ppy from e10.5, and finally somatostatin from e14.5 (170). Unfortunately, the expression of ghrelin was not addressed in the assays. Also regarding the initiation of the endocrine program, a sequential appearance of early endocrine lineage markers can be observed. In the dorsal pancreas, Neurog3 and Nkx2.2 expression is detected already at e8.75, then followed by Pou3f4 at e9.0. Pax6 can be detected at e9.25, and glucagon appears shortly after at e9.5. In the ventral pancreas bud, the order is the same but with a slight delay. Here, Neurog3 and Nkx2.2 appear at e9.5, Pou3f4 and Pax6 at e10.5, and glucagon and Ppy become expressed at e11.5. The role of Pou3f4 is unclear, and whether it is a marker for -cell progenitors or a marker for endocrine progenitors in general remains to be determined.

The detection of Neurog3-positive cells outside the Pdx1 domain and high-level expressing Nkx2-2 and Pou3f4 nuclei (it is unknown whether they are coexpressed or not) that are devoid of Pdx1 are unexpected observations suggesting that Neurog3 expression and endocrine lineage determination do not necessarily depend directly on Pdx1. Assuming that the high-level Nkx2-2-expressing cells are endocrine progenitor cells, an obvious question is whether they all, including those that seem to be devoid of Pdx1, depend on Neurog3 and controlled Notch signaling. Therefore, we have analyzed e9.0 Neurog3 and Hes1 mutants for Nkx2-2 expression and find that the amount of high-level Nkx2-2-expressing cells is profoundly up-regulated in the Hes1 mutants (Fig. 6B) and completely lost in Neurog3 mutants (Fig. 6C), showing that formation of cells with high-level Nkx2-2 expression indeed is dependent on Neurog3. This exemplifies the benefits of analyzing the whole embryo in contrast to sectioning because information about the whole organ regarding morphology and total immunoreactive cell numbers is provided. This allows analysis at the cellular level combined with a general overview of the morphological architecture. The raw data contain information on the entire pancreas that simplifies quantification and morphometric comparison between wild-type and mutant animals. It also opens the opportunity for preserving the entire data sets electronically and for the generation of databases. Along similar lines, a method that allows live imaging of pancreas morphogenesis was recently published (187). Here, real time images of branching pancreas morphogenesis and cellular dynamics were recorded using fluorescent markers. This technique complements the 3D imaging method applied to illustrate this review (17), and hopefully both tools will help to improve our understanding of the complex morphogenesis of the pancreas.

View larger version (17K): [in this window] [in a new window]   FIG. 6. Nkx2-2 is dependent on Neurog3 and controlled by Hes1. Collapsed image stacks of whole mount immunohistochemical detection of Pdx1 and Nkx2-2 in e9.0 wild-type (A), Hes1–/– (B), and Neurog3–/– (C) embryos. There is accelerated endocrine differentiation and Nkx2-2 expression in Hes1 mutants (B), whereas the cells expressing high levels of Nkx2-2 are lost together with the endocrine progenitor cells in mice lacking Neurog3 (C). However, when enhancing the signal it is possible to detect low-level Nkx2-2 expression in most of the dorsal Pdx1-positive cells (C, inset). Nkx2-2 is also seen in the ventral neural tube in all three panels where anterior is to the right, posterior is to the left, dorsal is above, and ventral is below.

	Note Added in Proof

Top Abstract I. Introduction II. From Endoderm to... III. Pancreatic Endocrine and... IV. Transcription Factor... V. Pancreatic Endocrine... VI. Pancreatic Endocrine Cell... VII. Summary and Future... Note Added in Proof References

 
Since the submission of this manuscript, Zhou and co-workers (188) have performed a lineage-tracing experiment where an inducible form of the Cre recombinase, controlled by the Cpal promoter, in combination with the R26R mouse was used to study the progeny of Cpal-expressing cells. They conclude that before e14.5, Cpa1 expression marks multipotent pancreatic progenitors that give rise to all three pancreatic cell types.

	Acknowledgments

 
We thank Hanne Duus Laustsen, Karsten Skole Marckstrøm, and Malene Jørgensen for technical assistance and Chris Wright, Helena Edlund, Catherine Tomasetto, Thomas Jessell, and Michael Rosenfeld for antibodies.

This work was dedicated to mark the 50th anniversary of The Hagedorn Research Institute.

Gene names comply with the official nomenclature (Mouse Genome Informatics).

	Footnotes

 
J.H.-S. is supported by the Juvenile Diabetes Research Foundation. O.D.M. and P.S. are supported by the National Institutes of Health (Grants DK072495 and DK072473), the European Union Sixth Framework, and the Juvenile Diabetes Research Foundation.

Disclosure Summary: The authors own equity shares in Novo Nordisk A/S.

First Published Online September 19, 2007

¹ M.C.J. and J.A.-R. contributed equally to this work.

Abbreviations: bHLH, Basic helix-loop-helix; BMP, bone morphogenic protein; Cpa, carboxypeptidase A; 3D, three dimensional or three dimensions; e9.5, embryonic day 9.5; ES, embryonic stem; FGF, fibroblast growth factor; Gcg, glucagon; Ghrl, ghrelin; Ins, insulin; Npy, neuropeptide Y; Ppy, pancreatic polypeptide; Pyy, peptide YY; RA, retinoic acid; Sst, somatostatin.

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