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Keywords:

  • pancreas organogenesis;
  • patterning;
  • tubulogenesis;
  • tip-trunk compartmentalization;
  • plexus remodeling;
  • branching morphogenesis;
  • endocrine specification and differentiation;
  • exocrine development;
  • human pancreas;
  • reprogramming

Abstract

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Pancreas oganogenesis comprises a coordinated and highly complex interplay of signaling events and transcriptional networks that guide a step-wise process of organ development from early bud specification all the way to the final mature organ state. Extensive research on pancreas development over the last few years, largely driven by a translational potential for pancreatic diseases (diabetes, pancreatic cancer, and so on), is markedly advancing our knowledge of these processes. It is a tenable goal that we will one day have a clear, complete picture of the transcriptional and signaling codes that control the entire organogenetic process, allowing us to apply this knowledge in a therapeutic context, by generating replacement cells in vitro, or perhaps one day to the whole organ in vivo. This review summarizes findings in the past 5 years that we feel are amongst the most significant in contributing to the deeper understanding of pancreas development. Rather than try to cover all aspects comprehensively, we have chosen to highlight interesting new concepts, and to discuss provocatively some of the more controversial findings or proposals. At the end of the review, we include a perspective section on how the whole pancreas differentiation process might be able to be unwound in a regulated fashion, or redirected, and suggest linkages to the possible reprogramming of other pancreatic cell-types in vivo, and to the optimization of the forward-directed-differentiation of human embryonic stem cells (hESC), or induced pluripotential cells (iPSC), towards mature β-cells. Developmental Dynamics 240:530–565, 2011. © 2011 Wiley-Liss, Inc.


OVERVIEW OF PANCREAS DEVELOPMENT

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

The pancreas is a compound gland that consists of two functionally and morphologically distinct cell populations derived from the endoderm. The exocrine pancreas consists of enzyme-secreting acinar cells arranged into clusters at the end of the ducts. Mature duct cells actively secrete bicarbonate and mucins, as well as having a more mundane plumbing function of draining acinar digestive enzymes towards the duodenum (reviewed in Slack,1995). The endocrine compartment of the pancreas comprises five different hormone-secreting cell types: the glucagon-secreting α-cell, insulin-secreting β-cell, somatostatin-releasing δ-cell, ghrelin-producing ϵ-cell, and finally the pancreatic polypeptide-secreting PP-cells. All of these hormones are involved in regulating nutrient metabolism and glucose homeostasis. The endocrine cells aggregate to form the islets of Langerhans, which are intermingled with blood vessels, neurons, and a mesodermally-derived stromal component. The intimate interaction between endocrine and vascular cells regulates hormone release, establishing a fine-tuned glucose homeostasis in the body (Slack,1995; Prado et al.,2004, Konstantinova et al.,2007).

Prior to organogenesis, the gut endoderm becomes grossly regionalized into distinct organ fields by a series of anteroposterior and dorsoventral patterning events, which are still far from understood. Generalizing from what is known about the patterning of other endodermal regions, such events are commonly mediated by extrinsic signals from adjacent mesodermal derivatives, as well as by intrinsic programs controlled by factors expressed within the endodermal cells themselves. In the case of the dorsal bud, the dorsal pancreatic region first receives inductive signals from the mesoderm during gastrulation, then permissive signals from the nearby notochord, dorsal aorta, and finally proliferative signals from the pancreas mesenchyme (Fig. 1A). Compared to the dorsal pancreas, the ventral pancreas is patterned by distinct sets of signals from the lateral plate mesoderm, cardiac mesoderm, and septum transversum (Fig. 1B).

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Figure 1. Early foregut endoderm patterning: signaling from adjacent mesodermal derivatives establishes the pancreatic domain. A: The early foregut endoderm is patterned by dynamic, distinct sets of permissive signals from the surrounding mesodermal derivatives. At E8, inductive signals (RA) from the paraxial mesoderm, together with suppression of Shh in the dorsal endoderm by FGF2 and Activinβ2 from the notochord, are required to establish the dorsal pre-pancreatic domain (left). At E8.5, dorsal aortae fusion pushes notochord away from dorsal endoderm. VEGF from dorsal aortae and vitelline veins (and even their earlier precursors, as shown in the ventral open foregut region, left) induce Pdx1 and Ptf1a expression in pre-pancreatic endoderm (middle). At E10, mesenchyme condenses around the budding pancreatic anlagen. FGF10 stimulates bud outgrowth and proliferation of the pancreatic progenitor pool (right panel). B: Sagittal view of an embryo at ˜E8 showing the specified dorsal and ventral pre-pancreatic endoderm (orange, top panel). Distinct sets of transcriptional regulators (blue text) and signaling molecules (red) control liver, ventral pancreas, and extrahepatic biliary (EHB) specification from common progenitors in the ventral foregut endoderm. Despite the dorsal and ventral pancreas sharing presumably highly similar genetic programs for cell differentiation, the gene activation cascade is different between these two anlagen, as depicted by the sequence of the listed transcription factors. Several of the primary (1°) pancreatic mutipotent progenitor cells (MPC)-specific transcription factors are maintained in secondary (2°) MPC.

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In the mouse, pancreas development is explicitly first evident at ∼E9.5 (Embryonic day 9.5) when the dorsal foregut endoderm thickens and evaginates as a relatively solid protuberance bulging into the surrounding mesenchyme. This is followed at ∼E10 by the emergence of the anlage of the ventral pancreas plus common bile duct, from the ventral foregut endoderm. Rodent pancreas formation has been classically divided into two overlapping waves of development. The first wave, referred to as the primary transition and occurring between E9.5–E12.5 in the mouse, is marked by a dramatic morphogenetic change of the pancreatic epithelium. The main events in this transition include active proliferation of pancreatic progenitors to generate a stratified epithelium, followed by formation of multiple microlumens and their subsequent coalescence, which serves as the precursor for turning the entire pancreatic primordium, via a plexus intermediate, into a growing epithelial arbor. The first differentiated endocrine cells, mainly glucagon-producing α-cells, begin to appear in the dorsal bud at this phase of development, in a cluster-budding process, rather than by individual delamination of epithelial cells (Pictet et al.,1972; Herrera,2000; Kesavan et al.,2009; Villasenor et al.,2010). At E11.5, the gut tube begins to undergo its first coiling movements, a rotation that brings the dorsal and ventral buds into proximity for their future conjoining into a single organ. By ∼E12.5, the densely packed epithelium starts to undergo active plexus remodeling, continued epithelial expansion and production of more plexus, sending finger-like protrusions into the mesenchyme, and over the entire organ. Here, it is important to realize a recently proposed alteration to the scheme of organ outgrowth: rather than a peripheral growth and epithelial extension via classical branching morphogenesis, it is now important (Fig. 2A) to consider that a complex process of plexus formation, proliferation, remodeling and further expansion, might afford growth and differentiation over the entirety of the organ; the process would include a massive amount of ‘outward pushing’ that arises from internal growth and remodeling.

Concomitant with these epithelial morphogenesis and remodeling processes, compartmentalization of the “protodifferentiated” pancreatic epithelium commences, segregating the epithelium into distinct “tip” and “trunk” domains. This distinction is now becoming defined better on the basis of molecular markers, including some of the transcription factors that are responsible for dictating the gene expression programs and fates of the cells in these regions. While the tip domains contain multipotential pancreatic cells (MPC), which are then believed to change into acinar-fated progenitors, the adjacent trunk epithelial region consists of an endocrine-duct bipotential progenitor pool (Zhou et al.,2007) (Fig. 2A). The new model for organ-wide plexus formation and remodeling provides a better link to a previous proposal that the number of the progenitor cells allocated to the pancreas primordium between E9.5–E12.5 determines the final size of the mature pancreas (Stanger et al.,2007).

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Figure 2. Overview models of mouse pancreas organogenesis. A: UPPER DIAGRAMS: Only dorsal bud shown for clarity. E9–E9.5: A region of cuboidal gut tube epithelium becomes pancreas-specified, forming a bulge of 1° pancreatic multipotent progenitor cells (MPC, red), which expands to form a stratified bud of several layers including outer cells that are basally polarized (lamina indicated by thick external outline in E11) and apically polarized inner cells that contact the central primary lumen. At ˜E11, individual cells within the body of the bud stochastically initiate apical polarization and constriction, which spreads to adjacent cells, forming “rosette intermediates” (for example, lower left panel) that present as multiple microlumens. Lumen coalescence (E12.5) and proliferation produces an epithelial plexus that is extensive at (E14.5), and now includes epithelial tubes proper. The plexus continues remodeling and grows further towards a final hierarchically organized, single tubular arbor (shown here at a later stage of differentiation/growth as “mature pancreas”). E12.5: Cells with nuclei indicate apically polarized, lumen-contacting cells that are future trunk epithelium (greenish), or retain MPC character (reddish) and are future tips (note: some are at non-tip locations). Remainder cells (those shown without nuclei) may be derived from outer or body cells of the preceding bud stage, as indicated by their relative shading; all cells will presumably join the epithelium eventually. E13.5–E14.5: During plexus formation/remodeling stages, MPC clusters are distributed broadly; those in “connectors” may become future tips. Substantial tip-splitting and lateral proliferation fuels rapid growth of the arbor during plexus growth and resolution (see Villasenor et al., 2010); “internal epithelial expansion” contributes substantially to overall organ growth. LOWER DIAGRAMS: highly schematic view of epithelium formation and tissue specialization towards acinar (red), or endocrine/duct lineages (blue, green, respectively), which emerge from the tip or trunk region. [Note: Plexus formation-remodeling is absent from lower diagrams to simplify presentation of patterning processes.] E10.75: A single rosette surrounding a microlumen is shown. Tip and trunk compartment specialization may begin just after, or concurrent with, tube formation (E12); early heterogeneity in MPC-character could facilitate tip-trunk organization. As above, tip regions are distributed broadly throughout the plexus epithelium. MPC that are distributed to forming tip regions (red) proliferate and undergo tip-splitting, causing outgrowth into the mesenchymal stroma and generating additional trunk epithelium with bipotentiality for endocrine (blue) or duct (green) fates. There may be some transient “softness” in the tip/trunk boundary regarding fate, indicated here by a transitional region with complementary red-to-green shading. Multipotency in tip regions is lost relatively quickly at ˜E13.5–E14.5, with cells in this location restricting to a proacinar and finally acinar state. Stage-dependent plasticity (shaded bidirectional arrows) may exist between tip-trunk character in cells at the border region before cells adopt stricter “tip” or “trunk/endocrine” identities. A specific intermediate cell state (not shown) at the border could represent the prospective centroacinar cells of the mature pancreas. “Mature pancreas” (top right) does not represent properly the proportions of acini, duct and islet (˜88%, 10%, 2%, respectively), but shows that acinar clusters (red) cap the termini of increasingly finely branched tubular ducts. Islets of various sizes throughout the pancreas comprise b-cells (blue core) plus other endocrine cell types (yellow mantle). B: Important transcriptional regulators expressed at each stage of pancreas development. −Pax4 and −Nkx2.2 indicate that e-cells develop in the absence of Pax4 and Nkx2.2.

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Starting at E13.5, the epithelium undergoes a striking morphogenetic transformation called the “secondary transition,” in that a massive differentiation wave and lineage allocation towards the three main pancreatic lineages occurs. Acinar cells arise from the extending tip epithelium and continue to undergo active proliferation to increase the number of acinar tips by duplication processes. The process of tip-splitting (Fig. 2A) can easily be seen to aid in explaining the rapid production of huge numbers of acini throughout the organ. Endocrine cells then become committed from endocrine-biased progenitors present in the trunk epithelial region (this region has also been historically referred to as “epithelial cord” or “primitive duct”). The delaminating endocrine cells go through a currently poorly defined epithelial exit process that is thought to involve an epithelial-to-mesenchymal transition (EMT) (Rukstalis and Habener,2007). Endocrine cells that leave the epithelium assemble into clustered endocrine islets but often stay in proximity to, even sometimes appearing relatively intimately associated with, one or the other of their parent ducts. It is still unclear if there is a classical asymmetric division of trunk endocrine progenitors that leads to one progeny cell entering an endocrine-committed state, with a relatively rapid basally directed exit from the epithelium, while the other daughter cell remains behind as a progenitor for future rounds of endocrine birth.

After the secondary transition, at ∼E16.5, the epithelium expands expands further, and this may be driven largely by acinar proliferation. Perhaps not surprisingly, the competence of the trunk epithelium to give rise to endocrine cells of various types changes according to the developmental stage. For example, β-cell competence arises coincident with the entry of the developing epithelium (Johansson et al.,2007). During late gestation and in the first few weeks of postnatal life, endocrine cells start to coalesce and round up into mature islets. In the mouse, predominantly β-cells form the islet core, and they are surrounded by a mantle of α, δ, and PP-cells, whereas the very minor ϵ-cell population (more of these cells exist during earlier organogenesis) is dispersed throughout the islet. About 80% of the islet cell mass present at birth is generated by the proliferation and differentiation of endocrine progenitors, with the other 20% coming from islet cell proliferation (Bouwens and Rooman,2005). After birth, the predominant mechanism for islet cell mass maintenance is self-duplication. There is a shorter refractory period for β-cell proliferation observed in the first four weeks postnatally and a longer refractory period after weaning (Bouwens and Rooman,2005; Teta et al.,2007; Salpeter et al.,2010). Studies on postnatal β-cell growth and proliferation have been reviewed extensively elsewhere and will not be covered in this review (reviewed in Ackermann and Gannon,2007; Bonner-Weir et al.,2010). In order to compensate for body growth, acinar differentiation, maturation, and proliferation also continue after birth before gradually decreasing until weaning (Desai et al.,2007; Salpeter et al.,2010).

To date, studies on the interplay between extrinsic signals and epithelium-intrinsic factors controlling pancreas organogenesis are still relatively sparse, especially on how cells move down a developmental pathway and progressively transfer to their final differentiation state from multipotential progenitors. A greater understanding of the cell biological and molecular mechanisms that regulate each of the developmental steps could be critical for implementing cell-replacement therapies for diabetes. It will certainly provide a proper way of validating the authenticity of any in vitro program of development, as well as provide clues as to how to direct cells down the desired forward differentiation track, or on how to partially rewind the developmental clock of mature cells to allow re-engagement of proliferation, or relax the mature cells' resistance to cellular plasticity. All of these features are relevant, for example, to finding clinically appropriate ways of restoring functional β-cell mass to diabetics, or of understanding the dysplasia and metastatic process in cancer.

We will review, comment, and speculate on each of these developmental processes sequentially, focusing our discussion mainly on mouse pancreas development, because of the greater depth of understanding of the developmental program in this organism. When known, we will note similarities and differences between mouse and other model organisms. We also present a direct comparison between human and mouse pancreas development in an independent section. At the end of the review, a brief discussion on current advances in pancreas regeneration/reprogramming and a perspective section on “How to unwind pancreas development within the next 5-10 years” are included.

PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Pancreas specification is presumed to result from early endodermal pre-patterning that leads to the establishment of tissue boundaries that roughly demarcate the regional allocations to specific organ primordia. Subsequent development of the presumptive pancreatic endoderm requires a series of permissive interactions with neighboring mesodermal tissues. There is a great need for more studies on the process of initial endodermal patterning, and particularly on when pancreatic specification shifts to a phase of hard pancreatic commitment; these issues are especially important to consider with respect to future work on the epigenomic regulation of the developmental process, as it could have direct implications with respect to how to induce plasticity and alterations of cell fates. As we shall cover below, the mesoderm produces various signaling molecules including FGF, BMP, Wnt, Retinoic Acid (RA), Hedgehog, and Notch that play dynamic and multiple stage-specific roles during endoderm patterning. Thus, precise spatiotemporal control of these tissue interactions is required for proper gut tube formation and pancreas organogenesis.

Broad Anterior-Posterior (A-P) Patterning of Endoderm

In the mouse, the different regions of the endodermal germ layer acquire a broad sense of their initial anteroposterior positional identity during gastrulation, when the endodermal cells migrate through the primitive streak. Cells exiting early go on to form foregut endoderm, while later exiting cells are allocated to the midgut and then the hindgut. The gut territories then become progressively refined, leading to specific organ locations. This process can be considered reminiscent of A-P segmental patterning in Drosophila, in which initially broad gap gene expression domains are established by threshold-dependent interpretations of pan-embryo gradients of transcriptional regulators; interaction between gap genes then cause progressive subdivisions, until individual segments become specified with particular fates.

A-P regionalization of the naive endoderm in the mouse embryo after gastrulation is established and refined by regional expression of organ-specific intrinsic factors induced by signaling molecules from the surrounding mesodermal derivatives, but how these interact with endoderm-autonomous regional cuing needs much more detailed study. There are several notable endodermal region- or organ-specific transcription factors (TFs) that are differentially expressed along the A-P axis, which include Six1 and Six4 (pharyngeal endoderm) (Zou et al.,2006), Sox2 (stomach) (Que et al.,2007), Nkx2.1 (lung) (Minoo et al.,1999), Hhex (liver) (Martinez-Barbera et al.,2000; Keng et al.,2000), Pdx1 (pancreas, duodenum, posterior stomach, common bile duct) (Offield et al.,1996), and the caudal homeobox genes Cdx1 and Cdx2 (intestine) (Beck et al.,1995,2003).

In mouse and chick, FGF4 from the mesoderm influences the anterior/posterior character of the newly formed definitive endoderm. There are reports that FGF4 acts directly on the endoderm by suppressing the anterior endodermal TF genes Nkx2.1 and Hhex, which are required for foregut endoderm formation, and to promote the expression of the posterior TF genes Pdx1 and Cdx1/Cdx2. FGF4 exerts its posteriorization effect on nascent endoderm in a concentration-dependent manner, and may therefore be a principal A-P domain-specifying influence from gastrulation to the early somite stages (Wells and Melton,2000; Dessimoz et al.,2006). But there is minimal to no information on how this influence is transduced at a transcriptional effector level. Similarly, Wnt/β-catenin signaling also has a posteriorizing effect on endoderm development. In Xenopus embryos, Wnt/β-catenin activity must be suppressed in anterior endoderm to maintain foregut identity and to allow liver and pancreas development, whereas high Wnt/β-catenin activity in the posterior endoderm promotes intestinal fate (McLin et al.,2007). Later it was shown that SFRP5-mediated suppression of both canonical and non-canonical Wnt activities (via inhibition of Wnt11) in the anterior endoderm is required for foregut specification and morphogenesis, while non-canonical Wnt activities in the posterior endoderm promote hindgut morphogenesis in Xenopus (Li et al.,2008). Determining the degree of parallelism in this process in the mammalian embryo will be important; it also has to be stated that much more precision on the timing and levels of signaling will be needed with respect to how specific organ territories are defined.

Retinoic acid (RA) is well known as a regulator of A-P patterning (reviewed in Marlétaz et al., 2006). With respect to the endoderm, RA may have a similar A-P regionalization influence in mouse, frog, zebrafish, and chick. In these organisms, RA from the overlying mesoderm posteriorizes the gut endoderm and promotes pancreas-organ allocation during gastrulation. Blocking RA signaling inhibits pancreas specification suggesting that RA is essential in this process (Stafford and Prince2002, Kumar et al.,2003; Chen et al., 2004b; Stafford et al.,2004,2006; Pan et al.,2007; Bayha et al.,2009). Based on the distribution of in vivo RA receptors (RAR) and results of cell-transplantation assays, it was concluded that RA acts directly on the endoderm for pancreas specification in zebrafish (Stafford et al.,2006). Studies in Xenopus, however, led to a different conclusion. Using mesoderm-endoderm recombinant explants and careful analysis of RAR isoforms distribution in vivo, it was demonstrated that RA acts both autonomously and non-autonomously on endoderm to elicit pancreas specification. For the non-autonomous effect of RA, RA-mediated prepancreatic endoderm patterning required parallel inhibition of BMP signaling and the presence of additional mesodermal signal(s) (Pan et al.,2007). One of the mesodermal signals might be FGF4, as it was shown recently to act synergistically with RA in chick endoderm A-P patterning (Bayha et al.,2009).

In zebrafish, Cdx4 expression limits the posterior boundary of the pancreas anlage territory, possibly by blocking RA signal transduction in the posterior endoderm. In contrast, the anterior boundary is restricted by Cyp26a1, which metabolizes RA and depletes its activity in the anterior endoderm, thus establishing the proper pancreatic field/size (Kinkel et al.,2008,2009). The level of conservation in determining pancreatic boundary establishment by Cdx4 and Cyp26a1 across vertebrate species has not been determined. In RALDH2 null mice, which lack a critical RA-synthesizing enzyme, the dorsal pancreas fails to bud and the early glucagon+ cells do not develop (Molotkov et al.,2005; Martin et al.,2005). This dorsal pancreas agenesis phenotype in RALDH2 null mice is reminiscent of the pancreas phenotype observed in Islet1 (Isl1) (Ahlgren et al.,1997) or N-cadherin (Esni et al.,2001) knockout mice. Because Isl1 expression in the dorsal pancreatic mesenchyme is also diminished in RALDH2−/− mice, this finding raises the possibility that the effects of RA (presumably its concentration) might be relayed via signals generated from Isl1-expressing dorsal mesenchyme. Failure of dorsal pancreas budding in RALDH2 mutants suggests that RA might play an instructive, rather than a permissive, role in pancreas specification in mice. As several genetic manipulations and mutants that are discussed later in this review (e.g., ectopic Sonic Hedgehog [Shh] expression, or Pdx1/Ptf1a double mutants) still do not block pancreas budding, there is the emerging feeling that initiation of pancreas budding seems remarkably “foundational,” and that the transcriptional regulation of dorsal pancreas “bud allocation and morphogenetic budding” deserves significant attention. In mouse and chick, RA is also required to maintain the pancreatic progenitors and to promote the commitment of these progenitors to Ngn3+ endocrine progenitors (Bayha et al.,2009; Oström et al., 2008).

Using swirl (BMP2−/−) and chordino (chordin−/−) zebrafish mutants, Tiso and colleagues (2002) showed that the dorsal-ventral BMP signaling gradient involved in the earliest steps of embryo body plan instruction is rapidly converted into instructions for the A-P patterning of endoderm in the developing zebrafish. They showed that Her5, which encodes a downstream mediator in Delta-Notch signaling, controls the translation of a BMP gradient into an anteroposterior regionalizing influence on the early zebrafish endoderm. In the absence of BMP2 signaling, Her5+ anterior endoderm is expanded and the pancreatic domain is reduced, which is opposite to that in chordino mutants. The authors propose that BMP and Delta-Notch signaling may act sequentially or in parallel to regulate Her5 expression, and that Chordin is required to inhibit BMP/Delta-Notch signaling dorsally to allow Her5 expression in the anterior endoderm (Tiso et al.,2002). In contrast to zebrafish, BMP inhibition is required for dorsal-ventral endoderm patterning and pancreas specification in Xenopus. Inhibiting BMP (via Noggin, or by misexpressing TGIF2, a homeodomain TF) establishes the pancreatic domain within Xenopus dorsal endoderm (Pan et al.,2007, Spagnoli and Brivanlou,2008). TGIF2 inhibits BMP/Smad1 signaling and selectively suppresses expression of a subset of TGFβ/Smad2 target genes in the dorsal endoderm during gastrulation, primarily through interaction with Smad1 and Smad2. TGIF2 suppresses liver formation by inhibiting Hhex expression while promoting Pdx1 expression (probably via direct binding to the Pdx1 promoter) to favor pancreas formation (Spagnoli and Brivanlou,2008). Later in this review, we will discuss how studies in the mouse revealed a possible resolution to these discordant findings, in that the level and dynamic nature of BMP signaling seems to be central to allowing these factors to have context/timing-dependent positive and negative influences on liver and pancreas specification from the ventral foregut endoderm.

DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

During the course of embryogenesis, the prepancreatic endoderm comes into contact with various mesodermal tissues, which function instructively and permissively in the specification, proliferation, differentiation, and morphogenesis of the pancreatic epithelium. As represented in Figure 1A, before budding occurs, the prospective dorsal pancreatic endoderm is closely associated with the notochord until E8.5, when fusion of the paired dorsal aortae displaces the notochord, bringing the prospective pancreatic endoderm into contact with vascular endothelium (reviewed in Slack,1995). By E9.5, condensation of the dorsal mesenchyme results in the evagination of the dorsal pancreatic rudiment (reviewed in Slack,1995). In the ventral endoderm, the pancreatic endoderm makes contact with cardiac mesoderm and the septum transversum mesenchyme (Rossi et al.,2001; Deutsch et al.,2001). Later, as the gut tube forms, the prospective ventral pancreatic endoderm comes into contact with a pair of vitelline veins (Fig. 1A). Thus, the extrinsic cues and genetic programs driving the formation of pancreatic tissue that emerges from the dorsal and ventral buds may be far from identical, and there are several mutations that result in the loss of one bud and not the other.

In vitro recombination studies on early chick endoderm showed that notochordal permissive signaling is involved involved in dorsal pancreas bud specification (Kim et al., 1997). These signals seem unlikely to be fully instructive because the notochord could not induce pancreatic gene expression in non-pancreatic endoderm (Kim et al., 1997). In mid-gestation mouse embryos, Shh is expressed at high levels in stomach and duodenal endoderm, but is excluded from pancreatic endoderm. The permissive signals from the notochord, mainly activin-βB and FGF2, might mediate Shh repression to allow pancreas bud formation (Hebrok et al.,1998) (Fig. 1A). Removal of notochord causes ectopic Shh expression in the dorsal pancreatic anlagen and perturbs dorsal pancreas development. This early block in pancreas development was probably a consequence of altering the dorsal pancreatic mesenchyme towards a more intestinal quality (Apelqvist et al.,1997; Kim et al.,1997; Hebrok et al.,2000). As will be discussed later, dorsal pancreatic mesenchyme is an important signaling tissue that allows proliferation and morphogenesis of pancreatic progenitors after the initial budding of the anlage. While inhibition of Shh seems to be required for early pancreas specification, later requirement for Hedgehog (Hh) activities has been identified in the expansion of the pancreatic epithelium, and in the regulation of insulin gene expression in the mature β-cells (Thomas et al.,2001; Lau and Hebrok,2010).

Somewhat confusingly, Hh signaling during gastrulation in the zebrafish seems to be required for the earliest steps of pancreas specification. Zebrafish with loss-of-function mutations in Shh (syu) or Smoothened (smo), as well as zebrafish embryos treated with cyclopamine (a hedgehog inhibitor), exhibit disrupted Pdx1 expression and endocrine pancreas does not form (Roy et al.,2001; diIorio et al.,2002). It was proposed that Shh from the notochord induces pancreas development in zebrafish through mechanisms that appear to mimic the late role of Shh in regulating amniote endocrine cell function. Furthermore, Shh signaling may also help in setting up anterior pancreatic boundaries by suppressing pancreatic gene expression in the anterior endoderm in zebrafish (Sun and Hopkins,2001; diIorio et al.,2007). There obvious is a need here to incorporate a proper analysis and consideration of the processes that lead to the formation of the principal and secondary islets, and how there may be repopulation of the initially formed principal islet by cells arising later from distinct progenitor pools. (Kinkel and Prince, 2009; Parsons et al., 2009; Wang et al., 2011). More recent studies More recent studies in zebrafish using lineage tracing and cell transplantation assays revealed that Shh signaling is required within adjacent endodermal cells for induction of pancreatic β-cells, via an intraendoderm interaction, during the stages of gastrulation and early somitogenesis (Chung and Stainier, 2008). One interpretation is that the apparent differences in the requirement of Hedgehog signaling during pancreas development in the zebrafish and amniotes might reflect differences in the temporal requirement for Hedgehog signaling, or that some of the effects might have been missed because most of the ex vivo studies analyses in amniote embryos were done at post-gastrulation stages. It remains an open question whether Hedgehog signaling from the midline mesoderm and/or the node is required during gastrulation, even if only for a short time, to initiate pancreas development in amniotes, as seems to be the case in zebrafish.

Signals from endothelial cells in the early dorsal aorta and vitelline veins seem to promote the initial stages of pancreas development. For example, aorta-less Xenopus embryos failed to express pancreatic genes, and co-culture of mouse dorsal prepancreatic endoderm with dorsal aortae could induce Pdx1 and insulin expression (Lammert et al., 2001). Furthermore, it was also shown that misexpression of VEGF-A (under a Pdx1 promoter) in transgenic mice caused islet hyperplasia and ectopic insulin+ cells in the stomach. By removing VEGF-A function in the Pdx1+ pancreatic epithelium, Lammert and colleagues showed that the endocrine cells signal back to the adjacent endothelium to induce a dense network of fenestrated capillaries within the islet, and that laminin/β1-intergrin from the vascular basement membrane is required for fine-tuning of blood glucose regulation (Lammert et al., 2003; Nikolova et al., 2006). Flk−/− (VEGFR2 knockout) mice, which lack endothelial cells, failed to activate Ptf1a expression in the dorsal pancreatic bud; while maintenance of pancreatic Pdx1 expression required interaction with aortic endothelium, despite the initial Pdx1 induction does not require these aortic endothelial signals (Yoshitomi and Zaret,2004). The aortic signals were later suggested to be relayed via the dorsal mesenchyme, by an effect of promoting the survival of dorsal mesenchyme, thereby maintaining FGF10 levels and inducing expression of the pancreas-specific “progenitor factor” Ptf1a and expanding the pancreatic progenitor pool (Jacquemin et al.,2006). Zebrafish cloche mutants that completely lack vascular endothelium have no effect on the early morphogenesis and differentiation of the pancreas even though the vascular endothelium is in contact with the pancreas during development (Field et al.,2003). It is possible that the pancreatic inducing signals provided by the endothelial cells in mouse and Xenopus are produced by different cell types in zebrafish; this hypothesis, however, would need to be addressed with precision.

VENTRAL PANCREAS INDUCTION

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

The lateral plate mesoderm (LPM) directly adjacent to the presumptive ventral pancreatic endoderm produces signals establishing the ventral pancreatic domain. With in vitro quail-chick tissue recombination assays, Kumar and colleagues (2003) showed that ventral prepancreatic endoderm receives instructive signals from LPM, and proposed that the pancreatic fate is specified at the 6-somites stage. Transplantation of already specified prepancreatic endoderm to a more rostral endodermal location did not abolish Pdx1 expression, while more caudal engraftment led to its re-specification towards a more caudal fate. Therefore, these instructive signals from the LPM, which might be BMP, RA, or activin, regionalize the endoderm in a posterior-dominant fashion (Kumar et al.,2003), the latter being a conserved theme in overall body plan patterning in many species.

Liver and ventral pancreas fates may diverge from an early common population of bipotential progenitors in the ventral foregut endoderm (Deutsch et al.,2001). It has been well studied in chick (Le Douarin, 1963) and mouse (Gualdi et al.,1996) that an endodermal interaction with cardiac mesoderm is required for proper hepatic differentiation. FGF signaling can induce hepatic differentiation in endodermal explants in the absence of cardiac mesoderm, while the prevention of FGF signaling from cardiac mesoderm makes ventral foregut endoderm unable to assume a hepatic fate (Jung et al.1999). Instead, pancreas fate is initiated, and has raised the perhaps debatable conclusion that the “default fate” of ventral foregut endoderm is pancreas (Deutsch et al.,2001). The septum transversum mesenchyme produces BMPs that are also necessary to induce a liver gene expression program and to allow some progenitor cells within a specific region of the ventral foregut endoderm to escape the pancreatic fate instruction. BMPs positively regulate the endodermal expression of GATA4, and act in parallel to cardiac-derived FGFs in the induction of liver fate (Rossi et al.,2001) (Fig. 1B).

A tissue-migration morphogenetic process is part of the mechanism that allows part of the ventral endoderm to avoid mesodermally derived hepatic-inducing signals. Blocking the relative movement of the ventral lateral foregut endoderm (the prospective ventral pancreas) prevents ventral pancreas specification from occurring. It was the genetic dissection of Hhex function in the mouse that led to this connection being made, with the tissue's “escape/repositioning” process being promoted by an Hhex-dependent proliferation of the leading edge of endoderm to allow it to rapidly extend away from the influence zone of the cardiac mesoderm (Bort et al.,2004). How extrinsic cues affect cell-fate specification needs to be considered as a highly dynamic, level- and context-dependent process. This concept was shown in an elegant study of the specification of liver-pancreas patterning by extrinsic signaling, wherein both inhibition and activation effects from BMP and TGF-β are carefully controlled over time, and can have distinct output effects (Wandzioch and Zaret,2009). Of course, it seems logical to presume that such ideas would also be relevant to a broader, earlier set of endodermal organ-patterning decisions, as well as into later stages when the intra-organ cell differentiation programs are being initiated and promulgated.

It has long been held that the extrahepatobiliary (EHB) system (including gall bladder primordium, extrahepatic bile duct, cystic duct) has its origin in the hepatic diverticulum. Recent studies, however, more strongly link the origin of the extrahepatobiliary primordium to the tissue that forms the ventral pancreas, rather than the liver. Sox17, a major regulator of endoderm formation (Kanai-Azuma et al.,2002), is required to establish and maintain distinct boundaries between the liver, EHB, and ventral pancreas domains. Loss of Sox17 function after gastrulation leads to biliary agenesis and ectopic pancreas formation, whereas Sox17 misexpression suppresses pancreas development by promoting ectopic biliary-like tissue within the posterior foregut region that expresses the posterior foregut and pancreas marker gene Pdx1. Sox17 upregulates Hes1 levels (another key player in EHB development, and well-known as a downstream mediator of active Notch signaling) in Pdx1-expressing endoderm; Hes1 then establishes a negative feedback loop to restrict Sox17+ biliary progenitors to the ventral foregut endoderm generating a boundary between EHB and ventral pancreas (Spence et al.,2009) (Fig. 1B). It is, however, not completely certain that Hes1 function in this context is definitively connected to Notch signaling.

Why vertebrates have dorsal and ventral buds with apparently distinct developmental histories, and if there is some type of selective function for islets derived from each bud, even in the mature organ, is a subject of (sometimes cryptic) conjecture, and of surprisingly few direct analyses. This is despite the potential usefulness of learning if ESC could be differentiated more rapidly and efficiently by choosing to push them along either a dorsal or ventral program, even if each track leads to similar and authentically functioning β-cells. At present, there is also a significant gap in our knowledge on the endodermal players that act in the time window between the early stages of endoderm formation and the regionalized expression of the posterior-foregut/pancreas marker gene Pdx1. A better understanding of endodermal regionalization over this period may explain how the endoderm is dynamically patterned to refine the presumptive organ domains. Development of more precise lineage-tracing tools and the ability to define prospectively the specific qualities of progenitor cells will in the future, hopefully, allow a deeper focus on the functional delineation between stages and mechanisms of specification, commitment, lineage allocation, and differentiation.

PANCREAS BUDDING AND THE PRIMARY TRANSITION

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

The first overt sign of dorsal pancreas budding is the thickening and evagination of dorsal midline endoderm caudal to the stomach at ∼E9, whereas ventral budding posterior to the liver is first evident approximately 12 hr later (E9.5). [In mouse, some report that two ventral pancreas “buds” are first formed, with one regressing during gut rotation (Lammert et al.,2001), in association with the process of the dorsal to ventral pancreas joining that occurs at ∼E12.5.] Shortly after E9.5, the buds grow out into the adjacent mesenchyme, forming essentially “solid buds” of stratified epithelium. This early pancreatic epithelium comprises mainly multipotent pancreatic progenitor cells (MPC) together with a few early-differentiated “first wave” endocrine cells, which are mainly glucagon+, although short-lived insulin+ and glucagon+/insulin+ cells were reported (Herrera,2000). Pulse-chase lineage-tracing experiments using Ngn3-CreER concluded that at least some of these first wave endocrine cells become incorporated into the mantle of the mature islets proper (Gu et al.,2002). The primary transition phase between E9.5–E12.5 generates an epithelial bud with appropriate progenitor numbers, which then undergo linked programs of proliferation, morphogenesis into a epithelial arbor, and extended programs of cellular differentiation along the duct, acinar, and endocrine routes. These processes lead to the growth and transformation into a correctly sized organ with a final tubular tree-like organ composed of ducts and acinar tips, with islets scattered between the branches (see Fig. 2A).

What controls the size of the pancreas? Stanger et al. (2007) produced evidence that the number of progenitors that are initially allocated to the pancreatic buds is correlated with the organ's final overall size: smaller numbers producing a much smaller organ with all cell fates still represented. The absence of a compensatory growth response to the reduced progenitor allocation suggests the possibility that the progenitors of all compartments (acinar, ductal, endocrine) are preprogrammed in some manner with respect to their future potential for division and differentiation decisions to be undertaken. In other words, size regulation is defined by an intrinsic program carried within these pancreatic progenitors. Stanger et al. (2007) discussed this type of organ size control as distinct from the “regulative programs” seen in other organs (e.g., liver, blood, central nervous system), in which reduction of progenitor numbers can be compensated by extensive proliferation, reduced apoptosis, with substantial effects from extrinsic factors (Conlon and Raff,1999).

EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

While the morphological partitioning of the early pancreatic buds as distinct dorsal and ventral buds might superficially suggest a significant movement towards the committed pancreatic state, the early pancreatic MPC may remain plastic for some time. For example, several genetic manipulations suggest that the early-stage bud cells can revert to an intestinal fate when faced with alterations in the carefully tuned intrinsic programs. As the buds grow, MPC progressively commit to the pancreas fate and move into a “protodifferentiated state,” a high-proliferation, epithelial expansion state that reflects the minimal expression of pancreatic-cell-type-specific differentiation products. MPC of the early pancreatic bud epithelium express several TFs associated with the initiation and commitment along a pancreatic program (Fig. 2B). Some of these regulators probably work actively in the regional allocation of pancreas fate within the primitive gut tube, while others promote progenitor proliferation and suppress differentiation during the primary transition. As many of these factors are also expressed in other endodermal regions, a familiar model once again arises in which combinations of TFs establishing the dorsal and ventral pancreatic domains. Similar to the extrinsic factors described above, these TFs play multiple (context-dependent) stage and lineage-specific roles during pancreas development. In accordance with models for heterologous organ development processes in several species, the spatiotemporal expression pattern of such TFs, and their levels, are tightly controlled in normal pancreatogenesis. Because most of the studies of the above TFs have been extensively reviewed elsewhere (Jensen,2004; Jorgensen et al.,2007; Oliver-Krasinski and Stoffers,2008; Gittes,2009), we focus here only on a few instructive examples, focusing on newly discovered roles in early MPC formation and maintenance, as well as highlighting some that are noticeably understudied and deserving future attention.

Mnx1

One of the best examples of a TF being required during several stages of pancreas development is the homeodomain protein gene Motor neuron and pancreas homeobox1, Mnx1 (also called Hb9 or Hlxb9). In the 8-somite stage (∼E8) mouse embryo, Mnx1 is expressed within the notochord and the entire dorsal and ventral endoderm at the prospective pancreatic level (Li et al.,1999; Harrison et al.,1999). Endodermal Mnx1 expression was reported to be transient and forming a dorsal-ventral gradient at E9.5 (Sherwood et al.,2009), with expression remaining in both pancreatic buds and the stomach until E12.5. More recent analysis (Pan and Wright, unpublished data) suggests that Mnx1 is not down-regulated but becomes redistributed to discrete pancreatic epithelium domains at E12.5, with its later expression becoming confined to Pax6+ endocrine precursors during the secondary transition. In adult pancreas, Mnx1 is expressed in mature β-cells (Li et al.,1999; Harrison et al.,1999).

Mnx1 expression in the dorsal pancreatic rudiment precedes Pdx1. Mnx1 null mice show dorsal bud agenesis and the normal Pdx1 upregulation during early bud formation does not occur, a phenotype that is stronger than the partial bud outgrowth seen in Pdx1−/− mice. The lack of dorsal bud formation in Mnx1 mutant mice is not completely understood. As Mnx1 is also expressed in the notochord, it may have non-autonomous functions on dorsal pancreas budding, relayed via notochordal signaling molecules. Because of the extensive alignment of notochord and dorsal endoderm along the A-P axis, notochordal Mnx1 expression may help establish a parallel pattern of its own expression broadly along the length of the dorsal endoderm. Furthermore, it is still unclear if Mnx1 function is required continuously for the expansion of pancreatic MPC pools, perhaps in some sort of mutually supportive interaction that reinforces expression of other MPC regulators, once budding occurs.

A different epistatic relationship between Mnx1 and Pdx1 exists in the ventral pancreas, because Mnx1 expression activates after Pdx1. In Mnx1 null mice, ventral pancreas development proceeds normally, producing all pancreatic cell types, but with reduced β-cell numbers and increased δ-cells. Those β-cells that do develop in the ventral bud are immature in that they lack Pdx1, Nkx6.1, and GLUT2 (Li et al.,1999; Harrison et al.,1999). We propose that further studies of the different functions of Mnx1 during the various phases of development, and the apparent variation between its role in the two buds are highly warranted.

Mnx1 over-expression, in Pdx1 promoter-driven transgenic mice, extending the high level of Mnx1 expression throughout the early pancreas epithelium, is detrimental to pancreas development (Li and Edlund,2001). This manipulation causes a large decrease in endocrine and exocrine cell differentiation, and the pancreatic mesenchyme seems to adopt a stomach/intestinal mesenchyme identity (Li et al.,2001). Mnx1 expression is higher in the prospective stomach compared to the pancreas anlage, suggesting differential threshold requirements for each organ (Pan and Wright, unpublished data), a phenomenon that could explain the trans-fating just described. Such a requirement for differential levels and temporal separation of function is also observed for Pdx1, as described below.

Pdx1

The Pdx1 gene can be considered as a central entry point for dissecting the complex signaling and transcriptional regulatory networks that orchestrate proliferation, outgrowth, and differentiation of the pancreas. It was the first gene shown to be cell-autonomously required for some of the earliest steps of pancreas formation, in mice and humans (Ohlsson et al.,1993; Jonsson et al.,1994; Offeld et al., 1996; Stoffers et al.,1997). Pdx1 is expressed in the dorsal and ventral pre-pancreatic endoderm at E8.5, and its expression is maintained at much later stages in the β-cells. In addition to almost complete agenesis of the pancreas, Pdx1−/− mutants have defects throughout the posterior foregut region, including distorted gastro-duodenal junction, loss of Brunner's glands, and defective enteroendocrine differentiation in the stomach and duodenum (Larsson et al.,1996; Offield et al.,1996; Jepeal et al.,2005). Later lineage-tracing showed directly that Pdx1-expressing progenitors produce acini, ducts, and endocrine cells of the mature pancreas (Gu et al.,2002).

Dorsal pancreas development arrests after initial budding in Pdx1 null mutants, resulting in a highly abrogated, developmentally retarded tissue, and a “ventral pancreas rudiment” cannot be found (Jonsson et al.,1994; Offield et al.,1996). Whether the ventral bud is formed initially but reverts to a bile duct or duodenal fate in the absence of Pdx1 function has not been elucidated. Significant numbers of first wave (immature) endocrine cells may still be formed in Pdx1 mutants, consistent with the notion that at least some of them form by an accessory Pdx1-independent program (Ahlgren et al.,1996; Offield et al.,1996). Using a tetracycline-regulated system, the deletion of Pdx1 function at E11.5 and E12.5 produced a cystic pancreas with very limited or no mature acinar formation, and diminished expression of the critical acinar regulator Ptf1a in such conditional mutants might be the direct cause of the phenotype. Therefore, maintaining the low level of Pdx1 expression in the acinar cells is required for their formation and differentiation (Holland et al.,2002; Hale et al.,2005). In acinar cells, Pdx1 forms a trimeric complex with Pbx1 and Meis2, which controls the nature of the transcriptional activity of PDX1 in acinar versus endocrine cells (Swift et al., 1998). The Pdx1-Pbx1-Meis2 trimeric complex cooperates with PTF1 to activate the acinar-specific elastase1 promoter (Liu et al., 2001).

The precise regulation of Pdx1 levels within the various pancreatic cell types (progenitors, transitional intermediates, and differentiated cells), and at different stages of specification and differentiation, are affected largely by the conserved 5′ cis-regulatory elements that represent sites of regulation for this gene by a plethora of trans-acting factors. There are four highly conserved regions, termed Area I–II–III (the “proximal enhancer region”) (Gerrish et al.,2000) and a more upstream Area IV (the “distal enhancer”) (Gerrish et al.,2004). Areas I–II impart islet-specific Pdx1 expression, while Area III working alone can confer β-cell-specific expression (Gannon et al.,2000). Areas I–II–III harbor binding sites for many TFs such as HNF1α, Pdx1 (Marshak et al.,2000; Gerrish et al.,2001), Foxa2 (Gerrish et al.,2000; Ben-Shushan et al.,2001), Pax6 (Samaras et al.,2002), HNF6 (Jacquemin et al.,2003), and MafA (Samaras et al.,2003). Deletion of the Area I–II–III cis-regulatory region creates a Pdx1 hypomorphic allele (Pdx1ΔI–II–III), and revealed that proper pancreas specification and outgrowth/differentiation from the foregut endoderm requires high Pdx1 levels. Low Pdx1 levels can drive largely normal development of many aspects of gut epithelium differentiation, such as gut/stomach enteroendocrine cell specification, structure of the gastro-duodenal junction, and formation of Brunner's glands (Fujitani et al.,2006, Boyer et al.,2006). Fujitani et al. (2006) also proposed the possible existence of different early progenitor populations (gut-proximal and gut-distal) in the dorsal pancreas bud, which have different developmental potentials when carrying the same level of Pdx1 expression from the Pdx1ΔI–II–III allele. Considering the relatively extensive deletion in the hypomorphic Pdx1 allele, these studies are considered relatively blunt. We are now aiming towards a higher resolution and more complete dissection, including TF-motif-specific deletions within Pdx1cis-regulatory elements, in order to build the most robust models of the gene regulatory networks underlying the intrinsic programs that govern the formation and behavior of various progenitor populations. Such studies should focus on cross-interactions amongst TF genes, the nature of regionalized signaling to gut-proximal and gut-distal regions of the dorsal bud, and how such signaling is established, dynamically regulated, and coordinated over space and time in forming endocrine and exocrine cell types from the endodermal epithelial arbor. With respect to bud MPC heterogeneity, it will be especially interesting to discover if one or another population has a higher potential to produce endocrine cells, as this sort of information might be applicable to in vitro directed differentiation of ESC/iPSC towards a cell-based therapy goal.

In comparison to the proximal Area I–II–III, the role of Area IV in Pdx1 expression is less clear. Recently, it was demonstrated that combined conditional Foxa1/Foxa2 null mutants in the Foxa3+ endoderm produced a more severe defect than Pdx1ΔI–II–III mutants, providing evidence that Foxa1 and Foxa2 regulate Pdx1 expression via Area IV, working in addition to the formerly reported Foxa2 site in Area III. Binding of Foxa1/Foxa2 to these regulatory elements is stage-dependent: Area IV seems to be occupied from early in pancreas formation, and is required for expansion and differentiation of pancreatic progenitors while Area III binding is linked more to the differentiation and function of mature β-cells (Gao et al.,2008). Whether Area IV is also required for progenitor expansion during early pancreas formation, either by itself or synergistically with Area I–II–III, demands more precise intervention such as spatiotemporally controlled Area IV specific deletion. These studies, nevertheless, suggest again that different threshold levels of Pdx1 production production have instructive value in directing lineage allocation decisions within the endocrine progenitor population, and/or promoting β-cell differentiation. Therefore, understanding the precise spatiotemporal and cell-type specific regulation of Pdx1 expression by transcriptional activator/repressor complexes acting via the various regulatory regions of the Pdx1 gene, and how they intersect with the epigenomic landscaping information, can be viewed as other avenues towards securing a broader knowledge base applicable in a translational direction towards the in vitro directed differentiation of hES cells, or the reprogramming of surrogate cell types.

Ptf1a

The trimeric Pancreas Transcription Factor 1 complex (PTF1) was first identified as an acinar enzyme gene activator (Rose et al.,1994; Krapp et al.,1996) and comprises a tissue-specific bHLH protein, Ptf1a; an ubiquitous bHLH protein, E2A or HEB (formerly known as p75, which provides a constitutive nuclear import function for the PTF1 protein complex) (Rose et al.,1994, Krapp et al.,1996, Sommer et al.,1991); and the distinct mammalian Suppressor of Hairless (RBP-J) protein, or the RBP-JL paralog (Obata et al.,2001; Beres et al.,2006). Ptf1a is expressed as early as E9.5 in most cells of the nascent pancreatic buds, and is essential for pancreas organogenesis in mouse and human (Kawaguchi et al.,2002; Krapp et al.,1998; Sellick et al.,2004). By recombination-based lineage tracing, it was demonstrated that Ptf1a is expressed in early pancreatic MPC, supporting specification of progenitors of acinar, endocrine, and duct cells (Kawaguchi et al.,2002). Ptf1a null mice lack the ventral pancreas, and dorsal bud outgrowth is rudimentary and extremely arrested. In addition to the complete lack of acinar cells, endocrine cell numbers are greatly reduced (Krapp et al.,1998). The remaining endocrine cells that are present include a significant number of β-cells that seem at least relatively mature as judged by their MafA+MafB state. Therefore, a certain proportion of the MPC population within the Ptf1a-null pancreatic buds seems competent to move forward and form relatively mature endocrine cells (Burlison et al.,2008). This leads to the premise that Ptf1a might be considered initially important for controlling the pancreatic fate acquisition by allocating sufficient MPC numbers to support the normal bud proliferation and outgrowth, rather than for the differentiation of endocrine cell types per se. This idea also fits with the finding that ductal epithelium outgrowth and differentiation are very limited in the Ptf1a-deficient situation (Krapp et al.,1998, Kawaguchi et al.,2002).

The lineage-tracing experiments of Kawaguchi et al. (2002), tracking cells that are instructed to activate the expression of Ptf1a as a marker of the initial push away from other organ fates and into pancreas specification, it was shown that Ptf1a-deficient MPC do not die but are effectively redirected to duodenum or common bile duct fates, the adjacent tissues from which the pancreatic buds erupt. A simple but important conclusion is that Ptf1a acts as an MPC organ-allocation switch factor, an early toggle between pancreas and posterior foregut endoderm states. More evidence for an essential role for Ptf1a in committing cells with the MPC competence to produce all pancreatic lineages come from studies in Xenopus embryos. Afelik et al. (2006) suggested that mis-expression of Ptf1a together with Pdx1 converts duodenum to pancreas, producing a “giant pancreas” that contained relatively normal acinar and endocrine proportions. One important role of Ptf1a after initial specification of the pancreas fate might be to ensure the rapid maintenance and/or upregulation of Pdx1 expression, possibly mediated by its transactivation via Area III of the Pdx1 promoter (Wiebe et al.,2007), with the idea being that Ptf1a/Pdx1 coexpression provides a more definitive pancreatic status to the MPC. Given these findings, it is pertinent to point out that a defined morphological dorsal bud forms even in Pdx1−/−;Ptf1a−/− double homozygous null mutant mouse embryos (Burlison et al.,2008). Thus, additional parallel-acting or upstream “progenitor-specification” factors must direct the initial allocation of endodermal cells to the pancreatic bud compartment. Or, more interestingly, perhaps the morphogenetic program of bud formation could be able to be uncoupled somewhat from the intrinsic programs of pancreatic specification and commitment.

The trimeric PTF1 exists in two “flavors,” varying according to the presence of RBP-J or its sister factor RBP-JL in the complex, which we here refer to as PTF1RBP-J and PTF1RBP-JL, and possess MPC or acinar-specific gene regulatory functions, respectively (Masui et al.,2010). During early stages of organogenesis, the RBP-J form is required for pancreas specification and progenitor maintenance. Pancreas development is greatly impaired, essentially to the same degree as in Ptf1a null mice, when a mutant Ptf1a protein is produced that carries a single tryptophan to alanine substitution near its carboxyl terminus that disrupts its ability to recruit RBP-J, but not RBP-JL (Masui et al.,2007). Consistent with this finding, humans with a C-terminal truncation of Ptf1a have pancreas agenesis (Sellick et al.,2004). RBP-J is a well-known major downstream activator of Notch signaling, although it might have additional functions and, indeed, it has been suggested that its role in early pancreas development as part of PTF1RBP-J is distinct from and independent of Notch signaling (Masui et al.,2007). Any conjecture that RBP-J carried within PTF1RBP-J complexes somehow limits the amount available for Notch signaling is hard to reconcile with the current model that active Notch signaling is one component of keeping cells in an undifferentiated condition (Murtaugh et al.,2003), the extrapolation being that there must be enough non-PTF1-bound RBP-J in the MPC if relatively widespread Notch activity is involved in keeping these cells in this state.

PTF1RBP-J complexes activate the expression of RBP-JL, and increased RBP-JL then begins to displace RBP-J, switching PTF1RBP-J to PTF1RBP-JL and initiating the acinar differentiation program (Masui et al.,2010). Since RBP-J and RBP-JL differ in transcriptional activity, the switch in TC-box-binding factor may cause an alteration in Pdx1 expression (and perhaps other genes that guide progenitor-to-differentiated cell transitions), from high levels in early MPC to the lower levels needed for acinar differentiation (Beres et al.,2006; Wiebe et al.,2007; Miyatsuka et al.,2007). These studies, together with the evidence above from frog embryo misexpression studies, point out the importance of positive cross-regulation between Ptf1a and Pdx1 in creating or maintaining the pancreatic “MPC metastable phase” as a priming phase for pancreatic progenitors in early stage organogenesis. During later lineage diversification, Ptf1a and Pdx1 become expressed at high levels in the distinct acinar and endocrine cell populations, respectively.

Although Ptf1a heterozygous mice do not show any obvious pancreas defect, gradual reduction in Ptf1a dosage in mice carrying a hypomorphic allele (Ptf1acbll/cbll) leads to pancreatic hypoplasia, truncated epithelial growth, severe abrogations in the extent of the epithelial arbor, and delayed acinar differentiation. The Ptf1acbll/cbll hypomorphic mice also develop neonatal diabetes as a result of the large loss of β-cell numbers, abnormal islet organization, and consequently insufficient insulin secretion (Fukuda et al.,2008). In contrast, studies from zebrafish carrying what is interpreted as another kind of Ptf1a hypomorphic allele, akreas, were interpreted as low Ptf1a levels promote endocrine fates, while high levels suppress endocrine fate and promote acinar differentiation (Dong et al.,2008). Nevertheless, the combined studies from mice and zebrafish are consistent in supporting the notion that differential Ptf1a levels, albeit together with cofactor variation, are associated with guiding pancreatic progenitor formation and maintenance, cell fate specification, and differentiation.

Sox9

Sox9 is a member of the SRY/HMG box (Sox) family, and was identified recently as involved in the proliferation, survival, and maintenance of pancreatic progenitors (Lioubinski et al.,2003; Seymour et al.,2007). This role is similar to its function in the hair bulge, intestinal epithelium, and neural crest (Cheung and Briscoe,2003; Blache et al.,2004; Vidal et al.,2005). Sox9 expression is first detected at ∼E10.5 in the early pancreatic MPC, and lineage tracing revealed that the “first-wave” Sox9+ progenitors, like Pdx1+ and Ptf1a+ progenitors, produce cells of all three pancreatic lineages (Akiyama et al.,2005). Sox9 expression persists in the endocrine/duct bipotent progenitor pool located in the central epithelial cords of the maturing epithelial arbor during the secondary transition (Seymour et al.,2007; Lynn et al.,2007). In the adult pancreas, Sox9 expression is maintained in a duct epithelium subpopulation, raising the possibility that it might demarcate some form of facultative progenitor pool. Seymour et al. (2007) showed that pancreas-specific Sox9 inactivation caused hypoplasia of both buds, through the decreased proliferation and increased cell death of progenitors, demonstrating that Sox9 is essential in MPC proliferation and survival. They also made links between Sox9 and the Notch effector Hes1 in maintenance of the progenitor cells. Human Sox9 haploinsufficiency causes abnormal acinar and endocrine pancreas formation (Piper et al.,2002). Studies by Lynn et al. (2007) reported that Sox9 regulates the expression of a battery of TF genes in the primary pancreatic MPC such as Onecut1/HNF6, TCF2/HNF1β, and Foxa2, perhaps implicating it as one of the central coordinators of the transcriptional networks that define the pancreatic MPC condition. Sox9 also regulates Ngn3 expression suggesting a role in initiation of endocrine differentiation (Lynn et al.,2007). The latter report also proposed that Sox9 might serve as a “Ptf1a switch cofactor” involved in toggling between the “MPC commitment” and “acinar-specification roles” of Ptf1a, since Sox9 was suggested to be capable of binding putative Ptf1a cis-regulatory genomic regions (Lynn et al.,2007). The nature of the crosstalk between Sox9 and Ptf1a is an important opening area, with much to be investigated.

HNF1β/TCF2

HNF1β, a MODY5 (Maturity Onset Diabetes of the Young 5) gene, is another regulator of endoderm patterning with an apparently potent role in pancreas progenitor expansion. HNF1β is expressed broadly through the foregut-midgut region at E8, and in the liver and both pancreas anlagens at E9.5 (Haumaitre et al.,2005). Solar et al. (2009) showed that the early HNF1β-expressing pool is multipotent and can seed all three compartments: acinar, duct, and endocrine. During the secondary transition, HNF1β expression becomes confined to the duct/endocrine bipotent progenitor domain of the central epithelial cord (the “mature trunk epithelium” described below), and is also duct-restricted in the postnatal or adult organ (Solar et al.,2009). In the absence of HNF1β, there is the transient formation of a dorsal pancreas ud that expresses Pdx1 and Mnx1, but these progenitors fail to expand. Similar to Ptf1a-null mice, the ventral bud is absent in HNF1β mutants (Haumaitre et al.,2005). The observation that Ptf1a expression is lost from HNF1β-null pancreas suggests the inability to enter the pancreas-specific commitment state, and that the absence of ventral bud specification is primarily via the loss of Ptf1a. The finding of HNF1β-binding sites on putative Ptf1a cis-regulatory elements suggests a direct regulatory relationship. As both Sox9 and HNF1β bind the Ptf1a promoter-enhancer regions, whether they cooperatively regulate Ptf1a expression to maintain the TF network and stabilize the progenitor gene expression programs by multiple (positive and negative) feedback loops is an attractive possibility that could be addressed by more precise experimental dissection. In addition to Ptf1a, HNF1β seems to form a transcription regulatory cascade with HNF6 in controlling pancreatic progenitor generation and endocrine progenitor induction (Maestro et al.,2003; Poll et al.,2006).

GATA4, GATA6

These factors are required in the endoderm induction process and for pre-pancreatic endoderm regionalization. Similar to HNF1β, Gata4 and Gata6 are initially expressed broadly over the early foregut endoderm, and later in the pancreatic anlagen (Decker et al.,2006; Watt et al.,2007). During the secondary transition, Gata4 and Gata6 expression becomes uncoupled and restricted to distinct pancreatic domains. Gata4 expression is restricted to acini between E13.5–E18.5. In the adult pancreas, Gata4 is expressed in a subset of α and β-cells and is no longer detectable in acinar cells. Gata6 mRNA is detected in the bipotential central epithelial cord of the secondary transition, but the pattern in later stages has not been characterized (Decker et al.,2006).

In Gata4 nulls, the ventral pancreas is not specified while the dorsal pancreas bud forms essentially normally. As Hhex is still expressed and the degree of proliferation seemed normal in the ventral endoderm in these mutants, the ventral pancreas defect was concluded as not caused by impaired morphogenetic migration of the ventral lateral foregut endoderm, thereby making it distinct from the Hhex mutant effect described above. Gata6 nulls exhibit a similar phenotype, although the phenotype is less severe, with a few detectable Pdx1+ ventral foregut cells compared to none in the Gata4 null (Watt et al.,2007). The mechanism underlying this differential response to Gata4 and Gata6 by the ventral and dorsal pancreas is unclear, but it is another example that highlights the difference in tissue interaction and genetic programs in the formation of each bud. In another experiment, transgenic mice with expression of Gata4/6-Engrailed fusion proteins in the Pdx1 expression territory exhibit complete absence of pancreas development, or reduced pancreatic tissue, and the conclusion was made that these GATA factors act cell-autonomously during pancreas development (Decker et al.,2006). The dynamic expression pattern of Gata4 and Gata6, moving into distinct tissue domains, suggests stage- and/or lineage-specific functions during later pancreas differentiation. Because of the early lethality of Gata4 and Gata6 null mice, deciphering these later functions will await pancreas-specific and temporally regulated deletion studies.

NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

The inference from studies manipulating Notch signaling is that the spatiotemporal regulation of this pathway is critical for preventing the precocious differentiation of all MPC into stage-dependent “default” programs, and that this “delay process” allows the coordination of epithelial outgrowth and proliferation with the timely production of endocrine and acinar cells, at the same time as allowing the organ to reach its destined size. The canonical view of the underlying mechanism is that Notch signaling controls whether cells proliferate as progenitors or differentiate, via a “suppressive maintenance” mechanism (Norgaard et al.,2003; Hart et al.,2003). Later, Notch signaling is a key regulator of binary cell fate decisions in endocrine specification, where it may use the classical “lateral inhibition” mechanism (see Endocrine Specification section). Multiple Notch ligands and receptors, as well as the mediator RBP-J and downstream target Hes1, are expressed in the early pancreatic epithelium. These include Notch1-4, Delta-like 1 (Dll1), Dll3, Jagged1, Jagged2, Serrate1, and Serrate2 (Lammert et al.,2000; Jensen et al.,2000b). Mice deficient for various Notch pathway components, such as RBPJ, Dll1, and Hes1, display pancreatic hypoplasia resulting from precocious endocrine differentiation that causes progenitor pool depletion (Apelqvist et al.,1999; Jensen et al.,2000b; Fujikura et al.,2006). Furthermore, overexpressing Notch3ICD, which represses Notch1-mediated upregulation of Hes1 in vivo, also results in reduced pancreas size and epithelial branching, with concurrent accelerated endocrine differentiation (Apelqvist et al.,1999). Conversely, mice expressing a constitutively active Notch1 Intracellular Domain (N1ICD) over the Pdx1+ endoderm have greatly impaired endocrine and exocrine differentiation, and the hypoplastic pancreatic epithelium is effectively trapped in the undifferentiated state appropriate to when the NICD expression is activated (Hald et al.,2003; Murtaugh et al.,2003). Collectively, these studies all lead to the conclusion that early Notch signaling is required for maintenance/expansion of early MPC, to allow a more regulated differentiation process in association with ongoing epithelial expansion.

A somewhat confounding recent study (Nakhai et al., 2008a), however, reported that deleting the two major Notch receptors, Notch1 and Notch2 from the early Ptf1a+ epithelium, had no effect on pancreas development. This rather surprising contradiction of the previous conclusions on how Notch works in progenitor maintenance could be explained if the expression of Notch3 and Notch4 were to be upregulated so as to compensate for the loss of Notch1/Notch2. Alternatively, Notch signaling in the pancreatic mesenchyme (which would not be inactivated using epithelium-specific Cre drivers) might also play a role in early pancreas development in addition to Notch epithelial activity. Consistent with this notion, Notch3 and Notch4 are expressed in the pancreatic mesenchyme during pancreas budding (Lammert et al.,2000; Jensen et al.,2000b). Such distinct epithelium/mesenchyme Notch functions were recently reported to control hair follicle differentiation (Hu et al.,2010). Another possibility is that Notch signaling is not required for early pancreas development, with the phenotype observed in RBP-J or Hes1 null mice representing loss of Notch-independent functions of these TFs. A Notch-independent function of RBP-J in the PTF1 complex and progenitor pancreatic allocation and maintenance was discussed above. It seems, therefore, plausible that the hypoplastic phenotype caused by epithelial N1ICD over-expression is explained by competition between N1ICD and Ptf1a for RBP-J, thereby blocking MPC allocation and/or proliferation, leading to stalled progenitor expansion. Non-canonical Notch signaling mechanisms have also been proposed to function in endocrine fate allocation. Deleting both Presinilin1 and Presinilin2 (the catalytic core in γ-secretase), as well as Notch2, in Ngn3+ endocrine progenitors results in Ngn3+ progenitors defaulting to an acinar fate, which expand rapidly to form the majority of acini compartments (Cras-Meneur et al.,2009). These rather suprising data have shed light on an interesting angle of Notch signaling in endocrine specification. Further biochemical analysis on the Presinilin1/2 and Notch2 cKO mutants, such as determining the proportion of Ptf1a-RBPJ versus N2ICD-RBPJ complex in wild type and Notch2 cKO mutants, might begin to test the hypothesis raised above. We note that a Notch-independent function of Hes1, although not directly tested in the pancreas, was proposed recently in retina progenitor cell proliferation (Wall et al.,2009).

POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

The pancreatic epithelium undergoes complex morphological changes during early development. Starting from a simple bud, the epithelium moves through a transient stratification, followed by polarity reacquisition and microlumen formation (Fig. 2A). Finally, microlumens fuse to form a luminal plexus that will later remodel into a complex tubular network (Kesavan et al.,2009; Villasenor et al.,2010). The extrinsic and intrinsic programs governing budding, progenitor expansion, and maintenance are arguably somewhat well-studied, but the cell biological aspects and molecular mechanisms driving tubulogenesis, and how tubule formation influences cell-fate specification, have only recently begun to be revealed. The recent alterations of models for epithelial formation in the pancreas will have ramifications on cell differentiation programs for years to come. Tubulogenesis in the pancreas shares similarities with salivary and mammary glands, in which a tubular arbor arises when groups of unpolarized epithelial cells undergo polarization to form microlumens, which then fuse to form a larger tubular network. This is distinct from the process that occurs in the lung, in which tube formation involves reiterative sprouting and stereotypical branching of a tubular anlagen consisting of fully polarized epithelial cells.

Initially, the pancreas consists of a bud of unpolarized epithelial cells surrounding a primary central lumen (PCL). The only implied polarity would be in cells contacting the basement membrane encasing the epithelial buds or those lining the PCL (Fig. 2A). Tube formation starts at E10.75 when apical polarization of single cells occurs stochastically in the bud. Targeting apical-membrane-protein secretory vesicles to the prospective apical surface polarizes these isolated cells, and the newly formed apical surface spreads across to neighboring cells, such that they begin to establish a common microlumen. These polarized epithelial cells undergo dramatic cell shape changes and rearrangements, forming “epithelial rosettes” that rather rapidly produce proper lumens at their common apical surface (Fig. 2A). Shortly thereafter, the microlumens expand and eventually fuse, converting the epithelium through a multi-lumen intermediate into a complex epithelial network or plexus. The forces driving the remodeling of the plexus into a classical epithelial arbor, which could involve both pruning and extension just as in the formation of blood vessels, should now be able to be investigated. Indeed, commonalities might be found with the process of vascular plexus remodeling, which occurs in several tissues during embryogenesis and is essential for producing the proper blood flow system. The end-result is functional single-lumen tree-like branches that ramify into the surrounding pancreatic mesenchyme. We would suggest that the formation and remodeling of the plexus intermediate, and the proposed large amount of tip-splitting-mediated epithelial expansion (Fig. 2A), must be studied in much more detail, via real-time and other high-resolution studies, with respect to several issues, including: (1) the location of cells with multipotential character, (2) the possible role of planar cell polarity signaling in generating a single tubular epithelial arbor from the plexus, which could well involve convergent-extension processes (making thinner, longer tubes from a previous shorter, wider form via realignment-intercalation of epithelial cells).

Tubulogenesis is a critical event during pancreas organogenesis because it is the tubular network that sets up the initial tip (future acinar location) versus trunk compartmentalization. Therefore, the process is fundamental to the appropriate development of requisite structures, and the spatially regulated deployment of various intercellular signaling systems, that together constitute the various niches for cell fate specification (Fig. 2A). With respect to the basic tip-trunk regionalization process, one idea is that progenitors that are in close proximity to the mesenchyme and receive a longer integrated exposure to extracellular matrix (ECM) acquire acinar fate, whereas progenitors receiving less mesenchyme exposure have a higher bias towards forming endocrine cells. The pancreatic tubulogenesis process has now been described in both zebrafish and mouse (Yee et al.,2005; Kesavan et al.,2009, Villasenor et al.,2010). Mapping the endocrine specification niches, and monitoring the density and location of endocrine commitment points throughout the remodeling epithelial plexus should provide clues as to the critical influences for the successful replication of this process in ESC being differentiated in vitro.

In one of these pioneering studies (Kesavan et al.,2009), altering Rho-GTPase Cdc42 function caused deficits in tube formation and cell fate specification. In pancreas-specific Cdc42 knockout mice, secretory vesicles were targeted apically but subsequent expansion of polarized surfaces to neighboring cells did not occur. Consequently, the lack of a common apical surface prevented lumen formation and tube establishment, and the unpolarized epithelium lost its integrity leading to epithelial fragmentation. The authors suggested that this defect led to increased exposure of epithelial progenitors to the surrounding mesenchyme/ECM, and promotion of acinar fates at the expense of endocrine cell numbers (Kesavan et al.,2009). Clearly, one of the most important foci for future investigation is how the formation of a highly organized epithelial tubular network through a plexus intermediate serves to provide the number, distribution, and type of niches that afford an effective pipeline for endocrine cell specification and commitment to the various hormone-expressing final cell states. It will be intriguing to find the intrinsic factors that induce Cdc42 action and trigger the stochastic single cell apical polarization in the pancreas bud. Whether or not microlumen initiation mainly occurs in the innermost regions of the early pancreatic bud, which receive lower input of extrinsic factors from the mesenchyme, such as FGF10, remains an open question.

EphB signaling plays an important role in several aspects of vascular remodeling during angiogenesis (Mosch et al.,2010), axon guidance (Bashaw and Klein), and neuronal patterning (Gelfand et al.,2009). EphB2/B3 receptors are also expressed in the pancreatic epithelium, and its ligands ephrin B1 and ephrinB2 expression are detected in the pancreatic mesenchyme (E12) and pancreatic arteries (∼E11.5), respectively (van Eyll et al.,2006; Villasenor et al.,2010). In the pancreas, EphB signaling is required for normal pancreatic epithelium morphogenesis, including microlumen formation. EphB2/B3 receptor mutants have disrupted epithelial rosette and microlumen formation, and delayed plexus remodeling, all resulting in a later-stage highly-deficient epithelial arbor. These mutants also produce less Ptf1a+ MPC at E12, which leads to an overall reduction of the endocrine/exocrine mass. The mechanism underlying the EphB mutants epithelial defects appears distinct from that in the Cdc42 mutants, and is probably due to altered epithelial adhesion, as both junctional β-catenin and E-cadherin expression are decreased in EphB mutants (Villasenor et al.,2010). This regulation of pancreas morphogenesis by epithelial-mesenchymal interaction of EphB2/ephrinB1 similar to Eph/ephrin regulation of vasculogenesis and angiogenesis (Oike et al.,2002), and provides another example of commonalities between pancreatic epithelial remodeling and vascular remodeling.

BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

During the period of approximately E12–E15, the tubular epithelial network expands and undergoes remodeling (as described in “Polarization and Tube Formation from the Protodifferentiated Epithelium” section and in Fig. 2A), in order to convert the proto-differentiated epithelium into a dynamic epithelium capable of generating the mature acinar and ductal cells, as well as providing the environment for the generation of the second wave of endocrine cells. Real-time imaging of the process has been reported for early pancreatic bud explants (E9.5 and E10.5, in which there are large numbers of MPC and the tip-trunk compartmentalization process is not complete; see Fig. 2A). It was proposed that ∼85% of branching in these early stages was via lateral branching mechanism, and only about 15% by terminal bifurcation (Puri and Hebrok,2007). It is important to remember, however, that in vitro culture methods for pancreas explants restrain their growth, and it will be useful to discern how these conclusions transfer to the 3D in vivo situation. It is possible that the relative fractions of the various branching modes change during embryonic organogenesis, and potentially even vary according to location within the growing organ. It is also possible that a combination of both branching modes might be used repeatedly, as observed for the lung (Metzger et al.,2008).

The cellular rearrangements and timing of plexus remodeling, and how this process leads to an epithelial framework that incorporates a systematic pattern of epithelial expansion (potentially with important spatiotemporal variations in the branching mode and/or density) could require a major re-evaluation of how early bud MPC undergo subdivision into the duct/endocrine versus acinar-fated progenitor pools. For example, how the simplified tip-trunk model (Zhou et al.,2007; Fig. 2A) fits into this emerging structure might require a large-scale, detailed 3-dimensional mapping effort, paying specific attention to the stage of pancreas development, and the general and precise geographic location within the expanding epithelial arbor. It seems inescapable that one must take a new view of organ expansion driven by internal and peripheral expansion, and not of classical branching morphogenesis. One noteworthy observation from the limited number of examples provided by Puri and Hebrok (2007) is that lateral branching seemed frequent near the extending tip epithelium, which could include cells that have substantial “retained MPC-like” character (see the following paragraphs in this section). This idea would be consistent with the ‘tip-splitting’ idea already raised above (Fig. 2A). Such branching would contrast the idea that lateral branching from within the trunk (within the endocrine/duct specification region) might involve some degree of rewinding of the cells' developmental clock to re-enter an MPC state in providing the new branch outgrowth point (that is, producing a new MPC-tip).

As already described, the implications from Villasenor et al. (2010) will be a major new influence here, and real-time monitoring of remodeling and branching with concurrent live mapping of various progenitor classes, transitional intermediates, and the committed/differentiating cell populations could provide massive insight. Another point raised by Villasenor et al. (2010) is the large expansion of the duct/endocrine “trunk epithelium” that they proposed to occur not by end-specific outgrowth extension, but by a process involving broadly distributed (both internal and peripheral) tip-splitting, lateral expansion and monolayer remodeling It is possible that the “MPC tip cells” in, for example, Zhou et al. (2007) were simply being focused on as the more peripheral population of these structures. A tip splitting-remodeling mechanism would be an attractive way of rapidly expanding the duct/endocrine containing “trunk epithelium” over the middle and later stages of pancreas organogenesis (including the secondary transition), while producing the massive numbers of future tip acinar-directed cells (we refer the reader to figure 2F in Villasenor et al.,2010).

Obviously, our understanding of pancreas branching is rudimentary, but these more recent reports open up rich areas for much deeper future exploration of how and when the niches that instruct cellular differentiation arise, and how the temporal window of competence to produce certain cells types might undergo closure. High-resolution real-time imaging and detailed in vivo quantitative genetic analysis (similar to that performed for the lung epithelium; Metzger et al.,2008) could provide dramatic discoveries related to the control of endocrine cell birth, and where, for example, β-cell versus other hormone-expressing cell fates are selected.

As shown in Figures 2A and 3, and as referred to above, the developing epithelial arbor can be considered to be composed of two functionally distinct compartments, rather loosely referred to as tip and trunk (and the reader should please note the impending modifications to this general model, as just reviewed). While the details still have to be worked out using precise marking and lineage-tracing techniques, one model is that the MPC in the early pancreatic buds rapidly become heterogeneous through the bud. Those cells maintaining the greatest degree of MPC quality are rapidly redistributed to the tips of the developing branched epithelium, either in association with or tightly following the microlumen formation process described above. The tip cells retain expression of the “primary multipotency markers” Pdx1 and Ptf1a, and have the molecular signature of Pdx1+Ptf1a+CpaI+cMycHI. Genetic lineage tracing studies performed using CpaI-CreER (here the enzyme gene Cpa1 could be considered a surrogate for Ptf1a, because Ptf1a regulates its expression) showed that tip cells at E12.5 produce all pancreatic cell types (endocrine, duct, acini), confirming their MPC nature (Zhou et al.,2007).

thumbnail image

Figure 3. Model of epithelial organization during the secondary transition. A single piece of tipregion epithelium from the secondary transition (˜E13.5) is depicted, consisting at this stage of two major domains: an MPC-containing tip domain (grey), and a trunk region harboring the endocrine/ duct bipotential progenitor pool (duct progenitors are shaded blue). The trunk domain is subdivided with respect to their age after birth from tip MPC. Trunk cells nearest the tip are youngest with older cells moving back down the trunk (teenage through oldest). Scattered cells within the trunk epithelium activate Ngn3 expression, with Ngn3LO cells (light pink) representing a putative metastable, relative plastic, uncommitted but endocrine-biased mitotic state. Ngn3LO asymmetric cell division leads to one daughter having higher Ngn3 expression (Ngn3HI: darker nucleus, red border) that becomes endocrine-committed, leaving a Ngn3LO progenitor available for more rounds of endocrine birth via production of additional Ngn3HI daughters. Committed Ngn3HI endocrine precursors rapidly activate Snail2 (purple cells) and escape the trunk epithelium, probably via epithelial–mesenchymal transition (EMT), before clustering to form the endocrine islets of Langerhans.

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For a limited time, tip MPC proliferation extends the arbor and drives remodeling of the epithelium, pushing the epithelial tips outward and leaving behind trunk-specified cells that possibly have a slower division rate (Zhou et al.,2007) (Fig. 2A). At ∼E14.5, tip MPC become depleted as they undergo PTF1RBP-J to PTF1RBP-JL -based switching to a proacinar fate (Masui et al.,2010) (Fig. 2B), consistent with lineage tracing showing CpaI-expressing cells at E14.5 only producing acini, not duct or endocrine cells (Zhou et al.,2007; Masui et al.,2007,2010). While this general principle has been developed using CpaI, it encodes a digestive enzyme that is hard to consider as an instructive factor for MPC formation or maintenance. Further lineage tracing analysis with additional genes, such as Ptf1a, may identify different populations of MPC that continue to exist within the epithelium after E14.5.

The central epithelial cord, or the “trunk” epithelial domain, contains a pool of cells with duct/endocrine bipotency (Solar et al.,2009), although it has still not been shown with rigor that individual cells are truly bipotent, in contrast to the mixed population premise (Fig. 3). Scattered cells within the trunk epithelium upregulate Ngn3 expression and commit to becoming endocrine precursors, which is generally associated with becoming mitotically quiescent. Endocrine precursor cells subsequently delaminate from the epithelial cord during the secondary transition. The timing of the choice to become a particular type of hormone-secreting cell, in reference to epithelial birth, delamination, and how this is regulated to ensure the appropriate colonization of the islets of Langerhans by the correct numbers of each cell type, remain mysterious and should definitely be an area of future profound discovery. The remaining non-delaminating HNF1β+Sox9+Nkx6.1+ trunk cells are fated to become duct cells (Seymour et al.,2007; Solar et al.,2009; Schaffer et al.,2010).

Irrespective of the details of the morphogenetic process, some information on the tip-trunk segregation process and relocation of MPC to the tip epithelium was recently suggested by Schaffer et al (2010), who proposed an early instructive cross-antagonistic relationship between Nkx6.1 and Ptf1a. The resolution of Nkx6.1 and Ptf1a expression into exclusive trunk and tip compartments, respectively, appears to correlate with the progressive restriction of trunk and tip cells towards the future duct/endocrine (trunk), or acinar (tip) lineages. This study further showed that Ptf1a is required for tip formation while trunk formation requires Nkx6.1 function, each factor being sufficient to repress the alternate lineage program (Schaffer et al.,2010). This type of mutually repressive relationship occurs in neural subtype specification in the spinal cord (Sander et al.,2000; Vallstedt et al.,2001), and it seems suitable to think of this “bistable switch” mechanism as applicable for several compartmentalization and/or lineage decisions during the entire process of lineage diversification from the pancreatic epithelium. Shifting the normal balance between tip and trunk domain can affect fate allocation: expansion of trunk domains generates more duct/endocrine cells, while increasing tip allocation leads to more acinar cells. The Nkx6.1/Ptf1a bistable switch operates during a critical competence window prior to lineage commitment, when progenitors are still multipotent. Direct transcriptional repression of Ptf1a by Nkx6.1 was proposed as one part of the mechanism [Nkx6.1 can bind putative Ptf1a regulatory elements (Schaffer et al.,2010)], although these conclusions were made from analyzing cell lines that are transformed, likely represent relatively mature endocrine cell types, and are therefore of unknown relevance compared to the embryonic cells that are actually engaged in the authentic program of compartmentalization. This possibility seems to require further support from, for example, analyzing the actively separating, or just-separated, populations of cells from the in vivo condition. Since Sox9 and HNF1β can also bind to the Ptf1a promoter, it will be interesting to examine if they cooperate with Nkx6.1 to suppress Ptf1a expression in the trunk domain. This type of mutual-antagonism relationship (between Nkx6.1/Sox9/HNF1β and Ptf1a) might be different from their early epistatic relationship in which positive feedback on transcription regulation amongst these TFs may be required to stabilize the pancreas fate, and for the expansion of the early MPC pool expansion (Haumaitre et al.,2005; Lynn et al.,2007).

In the Prox1-deficient pancreas, epithelial growth is disrupted with an absence of second wave endocrine cell production, concurrent with a large amount of precocious acinar differentiation. In addition to the role of Prox1 in maintaining the MPC pools by preventing acinar differentiation (Wang et al.,2005), these defects could perhaps just as likely be caused by imbalanced tip-trunk compartmentalization, indicating that Prox1 might be involved in establishing the proper allocation to the trunk compartment. Although tip-trunk compartmentalization starts at E12, as suggested in Figure 2A, the exact time window, what determines the sharpness of the boundary between the two domains, whether cells near the boundary can transit easily from one domain to another, or if there is a more plastic transitional zone, are all currently unknown.

“MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Over the years, it has been reported several times that post-budding epithelial proliferation, morphogenesis, and differentiation are dependent on interactions with the surrounding mesenchyme. In early in vitro cultures of E11 pancreatic bud (Golosow and Grobstein,1962), mesenchyme-free pancreatic epithelium failed to grow and differentiate, and recombination of “naked” epithelium with mesenchyme restored growth and morphogenesis. Because non-pancreatic mensenchyme also supported growth and morphogenesis of the epithelium in culture, this suggested that the mesenchyme-derived signals were permissive not instructive (Golosow and Grobstein,1962). Pre-”molecular genetic era” analyses of the requirement for the mensenchyme in pancreas development focused largely on its ability to promote epithelial branching and the appearance of the easily detected acinar zymogen granules. Later studies equipped with molecular markers for the analysis of the effect of the mesenchyme on the differentiation of various components suggested that the default fate of embryonic pancreatic epithelium is endocrine, and that this fate is redirected by mesenchymal signals to allow production of exocrine fates. For example, epithelial growth without mesenchyme in vitro, or when grafted ectopically under the kidney capsule, resulted in the formation of dense aggregates of pure islets with little or no acini or duct cells. By contrast, pancreatic epithelium co-cultured with mesenchyme resulted in predominantly acinar development, with fewer endocrine cells (Gittes et al.,1996; Miralles et al.,1998a). Miralles et al. (1998b) went further to show that follistatin, a normal product from the mesenchyme, could single-handedly push the production of exocrine cells at the expense of endocrine cell differentiation from the E12.5 rat (∼E11 in mouse) multipotent pancreatic epithelium.

The Fgf10/Fgfr2b pathway also functions in mesenchymal-epithelial crosstalk in the developing pancreas. During early stages (E9.5), the fibroblast growth factor receptor 2b (Fgfr2b) expression is detected in the epithelium, and its high affinity ligand Fgf10 is expressed in the pancreatic mesenchyme (Hart et al.,2003; Bhushan et al.,2001; Miralles et al.,1999). FGF1 and FGF7 are also expressed in the mesenchyme throughout pancreas development. As FGF10 expression diminishes at E11.5, these FGFs might be responsible for the later role of FGF in pancreatic epithelial branching morphogenesis (Miralles et al.,1999). Transgenic mice expressing dominant-negative Fgfr2b under the inducible metallothionein promoter, or mutants that lack either Fgf10 or Fgfr2b, display pancreatic hypoplasia (Celli et al.,1998; Revest et al.,2001; Bhushan et al.,2001). Furthermore, Pdx1 promoter-driven FGF10 mis-expression in the epithelium led to increased proliferation of pancreatic progenitors and a greatly reduced rate of differentiation of endocrine and exocrine cells. This effect of FGF10 again relates to the “suppressive maintenance” role of Notch in the pancreatic progenitors: excess FGF10 signaling leads to sustained Notch activation and promotes proliferation at the progenitor state (Norgaard et al.,2003; Hart et al.,2003).

Mesenchyme-derived BMP4/7, which signals through the epithelial ALK3 receptor, is also critical for pancreatic epithelium expansion and branching morphogenesis in mouse and chick. Inhibiting BMP signaling, either by over-expression of Noggin or a dominant-negative BMP receptor (dnALK3), causes severe pancreas hypoplasia, reduced epithelial branching, and excessive endocrine differentiation (Ahnfelt-Rønne et al.,2010).

While there are many studies focused on the mesenchyme-released signaling molecules, there are fewer studies focusing on the intrinsic factors that relay the effects of these intercellular, extrinsic factors. The LIM homeodomain TF Islet1 (Isl1) and the cell adhesion molecule N-cadherin, are both produced in the pancreatic mesenchyme during early pancreas development. Targeted disruption of either of these genes renders the mesenchyme unable to condense around the dorsal pancreatic bud. The resulting complete agenesis of the dorsal pancreas can be rescued by recombination of the mutant pancreatic epithelium with wild-type pancreatic mesenchyme (Ahlgren et al.,1997; Esni et al.,2001). The effect of N-cadherin loss on pancreas development seems to be secondary to a cardiovascular defect that causes diminished sphingosine-1-phosphate production from the endothelial cells. Sphingosine-1-phosphate, a circulating blood-borne factor, is required for pancreatic mesenchymal cell proliferation and survival. Its expression is restored upon cardiac-specific expression of N-cadherin, and can rescue the pancreas phenotype of N-cadherin-deficient mice (Edsbagge et al., 2005). While this indirect effect of N-cadherin via sphingosine-1-phosphate is required for proliferation and survival of pancreatic mesenchymal cells, the function of Isl1 in the dorsal pancreas mesenchyme remains to be determined; for example, what factors does it induce that are critical for bud initiation? A deeper insight into the mesenchyme-specific function of Isl1 will benefit our understanding of the interplay between Isl1 and the mesenchymal signaling regulating pancreas morphogenesis.

Across the early embryonic body plan, perhaps because of conditioning from the adjacent endoderm and more global patterning influences, the mesenchyme may carry much more region-specific character than previously considered. The physical movement of splenic mesenchyme away from dorsal pancreas mesenchyme is another key regulatory event in pancreas morphogenesis. This separation fails in null mutants for Bapx1 (Nkx3.2). Activin A from the mis-positioned splenic mesenchyme induces metaplastic transformation of pancreatic epithelium into gut-like structures, and disrupts pancreas morphogenesis greatly (Asayesh et al.,2006). This link between tissue movement with organ positioning and specification is reminiscent in some ways of the tissue movement problem found in the Hhex mutant, which was described above for the liver versus ventral pancreas fate decision.

THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

The secondary transition begins at ∼E13 in the mouse and represents a period of extensive epithelial expansion concomitant with a massive spurt of differentiation of endocrine, duct, and acinar cells. During this period, “second-wave” endocrine cells emerge from within the trunk epithelium, their nascent formation being marked by transient high levels of the pro-endocrine TF Neurogenin3 (Ngn3) (Gradwohl et al.,2000; Schwitzgebel et al.,2000; Jensen et al.,2000a; Gu et al.,2002). Following specification, Ngn3+ endocrine progenitors quickly differentiate into endocrine precursors, which delaminate from the epithelium and coalesce to form islets. Large numbers of β-cells start to appear and the α-cell population continues to expand during the secondary transition. It is believed that the majority of endocrine cells that contribute to the mature islets present at the end of gestation are generated during these stages. Proacini also begin differentiation from the numerous distal tips of the pancreatic epithelium, and move into synthesizing high levels of acinar digestive enzymes in addition to CpaI, such as amylase, elastase, and trypsinogen (Pictet et al.,1972). After about E14.5, acinar cells are mainly generated by the duplication of existing acinar cells, and endocrine progenitor birth in the trunk domain ends by the beginning of postnatal life (Zhou et al.,2007; Solar et al.,2009). Acinar cells and endocrine cells go through a maturation process lasting into postnatal life, and there is significant postnatal expansion of endocrine cell numbers, likely linked to self-replication (Desai et al.,2007; Finegood et al.,1995; Teta et al.,2007).

THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

As the epithelium expands, cell proliferation rates in the trunk region may be lower than that of the early tip-MPC (Zhou et al.,2007), but there is a need to maintain the trunk pool to ensure its replenishment and expansion even with the continuous delamination of endocrine-committed precursors from the trunk region [note that the tubercle splitting/expansion process proposed above (Villasenor et al.,2010) may be significant here]. For discussion purposes, we provide in Figure 3 a simplified tip-trunk model of the process of trunk epithelial endocrine specification, to act as a contextual framework to identify gaps in our understanding and discuss future directions for discovery. The general premise is that the trunk region is heterogeneous; in Figure 3, it is simply represented that a single, relatively long epithelial domain has some areas older than others, and not shown is the potential for regional heterogeneity in the types of mesenchymal-epithelial interactions engaged along the tube. Arguably, Figure 3 represents an “older model” derived from a canonical branching morphogenesis process, which does not take into account the Villasenor et al. (2010) report. In Figure 3, we suppose that trunk cells proximal to the tip domain are the youngest (having proliferated only once or twice), and older ones located distally have proliferated more times. Scattered cells within a specific “age-competence” region activate a low level of Ngn3 expression (becoming Ngn3LO), which represents a putative metastable, uncommitted but endocrine-biased, mitotic state. Ngn3LO cells may undergo asymmetric cell division and generate one daughter with higher Ngn3 expression (Ngn3HI) that becomes endocrine-committed, leaving a Ngn3LO progenitor available for more rounds of division and production of Ngn3HI daughters. A committed Ngn3HI endocrine precursor cell, and its immediate downstream transitional intermediates, rapidly activates Snail2 (purple) and escapes the trunk epithelium (probably involving epithelial-mesenchymal transition [EMT]), before clustering in the endocrine islets of Langerhans (Fig. 3).

In accordance with Villasenor et al. (2010), the plexus remodeling and tip-splitting expansion process would require a relatively modest rearrangement of this model to incorporate and consider the emergent models for epithelial organization, expansion, and maturation that are discussed briefly above, but the points made next are still relevant. There are several intriguing questions with respect to endocrine specification and formation. For example, it is not clear from the current tools and reagents how to identify, prospectively the first steps that distinguish the duct and Ngn3LO endocrine-biased progenitors from each other within the trunk epithelium. It is also unclear how many cell divisions each of the different classes of progenitors can undergo, and how proliferation of these classes is varied in accordance with the spatiotemporal needs to produce cells of the right number and type in order to produce the entire organ, and specifically the islets of Langerhans. Additional important characterizations will include how the remaining duct-specific progenitors in the trunk epithelium adjust their proliferation rate to accommodate the large number of cells that are presumed to leave the epithelium en route to the endocrine islet clusters.

ENDOCRINE SPECIFICATION

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

The current model of ductal and endocrine cell fate segregation, similar to the generation of neurons during neurogenesis, invokes Notch-regulated lateral inhibition. Under this model, committed endocrine progenitors expressing Ngn3HI autonomously induce the production of Notch ligands, which signal to neighboring cells and activate Hes1 expression. Hes1 in turn suppresses Ngn3 expression, and, thus, inhibits endocrine fate acquisition in the neighbors. It is still, however, uncertain if classical lateral inhibition causes single cells to become endocrine-committed, or if there is cluster-like acquisition of the Ngn3HI state and endocrine commitment. We caution here that conclusions on either side, depending on the analytical methods applied, could be erroneous if there is substantial cell mingling during the specification-commitment process. It is perhaps dangerous to assume that there are relatively static neighbor relationships, especially because the highly dynamic reorganizations of plexus remodeling and epithelial expansion could be associated with much cell shuffling in the trunk epithelium.

Ngn3 initiates the endocrine developmental program by inducing pro-endocrine TFs and factor(s) involved in delamination of the pro-endocrine cells from the epithelium. The direct targets of Ngn3 include NeuroD1/Beta2, Pax4, Arx, Insm1, Rfx6, Nkx2.2, Myt1, and Snail2/Slug (Huang et al.,2000; Smith et al.,2003,2004,2010; Watada et al.,2003; Mellitzer et al.,2006; Wang et al.,2007; Rukstalis and Habener,2007; Soyer et al.,2010) (Fig. 2B). The activation process for Ngn3 appears to take input from TFs such as Sox9, Hnf1β, Hnf6, Hnf3β, and Pdx1, for which binding of these TFs to the Ngn3 distal regulatory region has been detected (Lee et al., 2001; Lynn et al., 2007; Oliver-Krasinski et al., 2009). As already suggested, how these regulators control Ngn3 activation in a limited number of cells within the trunk epithelium demands much attention.

Mice lacking Ngn3 function fail to develop endocrine cells (Gradwohl et al.,2000), whereas ectopic Ngn3 expression under the Pdx1 promoter induces premature differentiation of the entire pancreatic bud progenitor pool into endocrine cells with predominantly α-cells (Apelqvist et al.,1999; Schwitzgebel et al.,2000). Lineage tracing studies showed that all endocrine cell types seem to be born from Ngn3-expressing endocrine progenitors (Gu et al.,2002). There is recent evidence showing that Ngn3 expression is maintained at low levels in mature β-cells, and that it has a function in them (Wang et al.,2009). Recent clonal analysis by Degraz et al. (2009) further revealed that the majority of the Ngn3HI endocrine progenitors are individually unipotent and mitotically quiescent, a conclusion in accordance with previous studies (Gu et al., 2002). That is, they are precursors of either an α-cell or a β-cell, but not of both at the same time, and each Ngn3HI cell generally gives rise to only one endocrine cell. They further suggest that Ngn3HI cells might have already been pre-committed or pre-biased to a specific endocrine cell lineage, but the timing of this selection-point needs much better analysis. A minor proportion of post-Ngn3 islet precursors divide, though only a few times, well into adulthood under physiological conditions (Jensen et al.,2000a; Degraz and Herrera, 2009). The remaining issue is how cells choose (or are instructed to choose) the proper/appropriate final hormone cell type; when is the choice taken with respect to the increase to transient Ngn3HI production, before or after, how is it controlled, and can we override these choices either in vivo or in vitro towards a therapeutic advantage?

One important finding has been that the stage and context within which Ngn3-expressing cells arise dictates their endocrine progenitor competence. Johansson et al. (2007) showed that the pancreatic epithelium moves through competence states that affect the output from Ngn3-expressing cells and that these changes in competence intrinsic to the epithelial cells themselves. Their “Ngn3 addback” strategy used transgenic mice with inducible Ngn3 expression under the control of the Pdx1 promoter in the Ngn3 null background, to demonstrate that early Ngn3+ progenitors (E8.75) produce α-cells, whereas endocrine progenitors acquire the competence to make β-cells and PP cells at ∼E11.5 and E12.5, respectively, with much increased competence towards the β-cell fate at E14.5. Endocrine progenitors become competent to produce δ-cells at E14.5.

The timing and level of Ngn3 are critical in driving entry into the endocrine lineage and fate allocation. By using hypomorphic alleles of Ngn3, Wang et al. (2010) demonstrated that the move to Ngn3HI was necessary for irreversible commitment of cells to the endocrine lineage. Cells with moderate to low levels of Ngn3 can avoid endocrine cell fate commitment and select other pancreatic cell fates. Consistent with this idea, by using a more sensitive lineage labeling tool that allows labeling of Ngn3LO cells (Ngn3-Cre BAC transgene with R26EYFP reporter), we ourselves have observed large numbers of Ngn3-lineage labeled cells, which are not Ngn3HI, present in the secondary transition trunk epithelium at E16.5 (Boyer and Wright, unpublished observation). In reference to Figure 3 and the more detailed discussion above, the general idea is that low levels of Ngn3 (and our lab has recently detected evidence for this behavior) pre-empt Ngn3HI; the Ngn3LO state represents a metastable, relatively plastic and uncommitted condition, and that elevating the levels of Ngn3 works like a one-way railroad switch to divert cells specifically down the endocrine pathway.

ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

While we do not know when the choice is made regarding which of the five endocrine subtypes an Ngn3HI precursor will attain, we are learning more about the “TF codes” that are specific for each cell type and the transcriptional regulatory networks controlling differentiation and maturation are becoming better defined. Major alterations in the allocation to the endocrine cell populations are caused by interfering with the cell-type-specification code, and in some cases the application of lineage-tracing studies has provided stimulating insight into the plasticity of the terminally differentiated endocrine cells. Many TFs have been identified to function in endocrine subtype lineage allocation, differentiation, and maturation. These TFs include Pdx1, Mnx1, Nkx2.2, Nkx6.1, Pax4, Pax6, Arx4, Foxa1, Foxa2, HNF4, Islet1, Insm1, Rfx6, MafA and MafB (see Oliver-Krasinski et al.,2008, for greater depth on individual factors).

For this review, we introduce classes of phenotypes observed during endocrine subtype specification and differentiation. In general, three classes of regulators may be divided, based upon our current understanding of the phenotype: (1) general endocrine differentiation (NeuroD1, Islet1, Insm-1, Rfx6, Nkx6.1), (2) lineage-allocation (Pdx1, Pax4, Arx, Nkx2.2), and (3) maturation factors (MafA, MafB, Foxa1, Foxa2, NeuroD1). Some of the TF mutants show more than one phenotype, suggesting multiple functions during endocrine development. Sequential triggering of all three classes of factors is necessary to generate the correct numbers of functional mono-hormone endocrine cells. The proper phasing of endocrine cell ontogeny occurring in the framework of a three-dimensional epithelial structure can be regarded as a tightly orchestrated pipeline for endocrine cell production that feeds the building blocks of the islets.

CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

General endocrine factors facilitate the formation of proper endocrine cell numbers by maintaining survival and/or stimulating proliferation of endocrine progenitors/intermediates. This class of factors also permits endocrine cells to move into the mono-hormone-producing cell state. Several TF genes that fall into this category [e.g., NeuroD1 (Huang et al.,2000), Insm-1, Rfx6, and Islet1] are downstream, primarily direct targets, of Ngn3. NeuroD mutant mice have arrested endocrine cell development during the secondary transition, with reduced numbers of all endocrine cell types. The remaining endocrine cells fail to form islets (Naya et al.1997). Such deficiencies are also observed in Insulinoma-associated antigen 1 (Insm1), Regulatory X-box binding 6 (Rfx6), and Islet1 (Isl1) mutants. Loss of Insm1 or Rfx6 arrests the differentiation of endocrine precursors towards hormone-expressing cells (Gierl et al.,2006; Mellitzer et al.,2006; Soyer et al.,2010; Smith et al.,2010). A phenotype similar to Rfx6 mutant mice has also been reported in human infants carrying mutation in the Rfx6 gene, who have an autosomal recessive syndrome of neonatal diabetes, Mitchell-Riley syndrome (Smith et al.,2010).

Isl1, apart from its initial role in pancreatic mesenchyme for dorsal pancreas induction, is also required for the proliferation and survival of post-Ngn3 endocrine cells during the secondary transition (Du et al.2009). Since Insm-1, Rfx6, and Isl1 expression are maintained in adult islet cells, determining any remaining later functions in maturing or adult islet cells awaits a temporally controlled cell-type specific knockout.

Nkx6 family TF genes (Nkx6.1 and Nkx6.2) could be the only non-Ngn3 direct target that fall into this class. The Nkx6.1 null has substantially reduced β-cell numbers while both β and α-cells are reduced in Nkx6.1/Nkx6.2 double null mice, demonstrating an intriguing overlapping yet distinct requirement for the different Nkx6 TFs. The number of Ngn3+ endocrine progenitors and islet precursors are reduced dramatically in these mutants, suggesting that Nkx6 might act upstream of Ngn3. These studies also proposed that Nkx6 factors promote β-cell fate specification from Pdx1+ progenitors and that Ngn3 subsequently promotes cell differentiation by initiating cell cycle exit, in association with the not-yet-understood process of inducing the lineage-specific choice (Henseleit et al.,2005). The latter idea could be considered controversial. We, however, suggest a different possibility, in that Nkx6.1 function might be required to establish the proper trunk epithelium size/domain or structure prior to the actual endocrine specification event, because this would subsequently affect the number of endocrine progenitors generated from this domain. This idea would be consistent with the cross-antagonism and bistability switch relationship proposed between Ptf1a and Nkx6.1 during the compartmentalization process (Schaffer et al.,2010), as discussed above.

CLASS II: LINEAGE ALLOCATION FACTORS

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Lineage-specific factors control the flux of endocrine progenitors towards a specific endocrine type. As a consequence of removing such factors, endocrine cell numbers are largely unaffected, but fractional islet allocations are often severely altered. Nkx2.2, Pax4, Arx, and Pdx1 mutants highlight the extreme lineage-switching caused by such single factor removals. Total endocrine cell number is almost normal but the majority of the β-cells, and a substantial fraction of both α-cells and PP-cells are replaced by ghrelin-producing ϵ-cells in Nkx2.2 mutant mice (Sussel et al.1998; Prado et al.2004). In Pax4 null mice, β and δ-cells are lost concomitant with a remarkable increase in α-cell and ghrelin+ ϵ-cell numbers. The increased ϵ-cell number is similar to the Nkx2.2 null pancreas, suggesting possible genetic interactions between Pax4 and Nkx2.2 (Prado et al.,2004).

Pax4 also directly inhibits expression of Arx, which encodes a homeodomain TF that is a key α-cell specification factor (Collombat et al.,2003,2005). A mutually repressive relationship between Pax4 and Arx is evident by a lineage switch in Arx mutants — a loss of α-cells and concomitant increase in β/δ-cells — that is complementary to the alteration in Pax4mutants. Furthermore, Pax4 and Arx induce, respectively, either an α-to-β or β-to-α conversion upon misexpression in terminally differentiated mature endocrine cell types (Colombo et al.,2007; Collombat et al.,2009). Thus, Pax4 and Arx are required for β and α-cell fate allocation, respectively. Although Pax4 null mice also lack somatostatin+ δ-cells, a positive selective role for Pax4 in δ-cell development remains unclear. In addition, Pax4/Arx double null mice show a massive increase in δ-cells, which suggests that δ-cell terminal differentiation requires the absence of Pax4 and Arx (Collombat et al.,2005).

Pdx1, apart from its early roles in MPC outgrowth and expansion, is also required for β-cell proliferation and survival. Deleting Pdx1 function specifically in β-cells leads to a dramatic reduction in β-cell number concomitant with α/δ-cell overgrowth. Lineage tracing analysis confirmed that these α-cells and δ-cells do not come from β-cells, rather their increased numbers result from increased proliferation. Thus, this study proposed that one of the β-cell functions is to inhibit proliferation of adjacent islet cell types, and that endocrine sub-type intercommunication is required to generate the appropriate proportions of the various endocrine subtypes (Gannon et al.,2008).

CLASS III: MATURATION FACTORS

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Maturation factors seem to control later aspects of the final move towards physiological readiness, although in some cases there may be a degree of overlap with a harder to decipher role in the preceding phases of lineage allocation and commitment. Loss of these factors causes little or no defect during endocrine cell lineage diversification (at least that is understood at the current level of detail), but the cells begin to show aberrant function postnatally or in adulthood. The bZIP factors MafA/B are expressed relatively later in development and are essential to acquire and maintain the mature state of hormone-expressing cells by activating genes important for β-cell function. These genes include insulin, Pdx1, GLUT2, Nkx6.1, Slc30a8 (zinc transporter) and G6pc2 (Glucose-6-phosphatase catalytic sub-unit 2 protein) (Aramata et al.,2005; Zhao et al.,2005, Raum et al.,2006; Artner et al.,2007). During development, β-cells move from a MafB+ immature state to MafA+/MafB+, and finally, to a MafA+ mature state. Thus, MafB is essentially absent from mature adult β-cells and its expression is restricted to α-cells in the adult pancreas. The switch from MafB+ to MafA+ in β-cells occurs during a period of β-cell maturation and this process is associated with up-regulation of Pdx1 expression (Nishimura et al.2006; Artner et al.,2010). MafA mutants have no effect on endocrine cell development but the mice become glucose-intolerant postnatally (Zhang et al.,2005). Whereas MafA function is β-cell specific, MafB is required for both α-cell and β-cell differentiation/maturation, and is a more potent regulator of embryonic β-cell development than MafA. MafB−/− mice have reduced α and β-cell numbers, as well as some form of apparently delayed β-cell development (Artner et al.,2006,2010).

Expression of Foxa2 begins very early in the definitive endoderm, and persists throughout the early pancreatic epithelium. Conditional inactivation of Foxa2 (Foxa2-cKO) in early pancreatic epithelial precursors does not affect MPC development but leads to late-arising defects in α-cell terminal differentiation. This result might be due to functional redundancy in the early phase with the related Foxa proteins, Foxa1 and Foxa3, which is supported by the observation that early pancreas development is severely perturbed in pancreas-specific Foxa1/Foxa2 double knock-outs as a result of loss of Pdx1 expression (Gao et al.,2008, and see previous discussion above). Such functional overlap has helped in uncovering later aspects of Foxa2 function. Initial specification of α-cells in the Foxa2-cKO proceeds normally but differentiation is blocked with 90% reduction of glucagon expression and complete lack of prohormone convertase 2 (PC2) production in these mutant mice (Lee et al.,2005). Mutant mice with specific ablation of Foxa2 in fetal β-cells have disorganized islet architecture and dysregulated insulin secretion as a consequence of reduction in the expression of KATP channel, Kir6.2, and sulfonylurea receptor 1 (SUR1)]. The resultant neonatal Foxa2 mutants die shortly after birth from severe hyperinsulinemic hypoglycemia (Sund et al.,2001). These findings on the Foxa2 requirement in fetal endocrine cells were added to by recent studies indicating that Foxa2/Foxa1 maintain the normal physiological status of the mature pancreas. A late-stage deletion in mature β-cells of Foxa2 or both Foxa1/Foxa2 resulted in increased insulin-granule docking and exocytosis, which is the main cause of persistent hyperinsulinemic hypoglycemia. Thus, these Foxa proteins control multiple aspects of insulin secretion in mature β-cells, and regulate many genes involved in the glucose sensing, metabolism, and granule exocytosis machinery (Gao et al.,2007,2010).

The mesenchymal role for N-cadherin discussed earlier in this review is distinct from another function, in which N-cadherin is involved in controlling insulin granule turnover and secretion when its expression later becomes confined to islet cells. Deleting N-cadherin in the Pdx1 expression territory results in reduced insulin granules and impaired insulin secretion in β-cells. This suggests that cell adhesion or perhaps coordinated β-cell communication amongst the population is critical for proper regulation of insulin granule turnover (Johansson et al.,2010).

A late-stage maturation role for NeuroD1 in β-cells was recently proposed by Gu et al. (2010). When NeuroD1 was specifically inactivated in mature β-cells, the NeuroD1 null islets responded poorly to glucose and displayed a glucose metabolism profile similar to immature β-cells, with the key features of increased expression of glycolytic genes and lactate dehydrogenase (LDHA), elevated basal insulin secretion and oxygen consumption, and neuropeptide Y (NPY) overexpression. Furthermore, KATP channel-mediated insulin secretion was defective. Therefore, NeuroD1 is required for the complete transition to β-cell maturity, and maintenance of full glucose-responsiveness (Gu et al.,2010). Another noteworthy observation was that virtually all insulin produced by the mutant β-cells was exclusively derived from Ins2, with Ins1 expression very diminished. These features could represent a useful indicator of β-cell maturity status, allowing the distinguishing of immature from mature β-cells generated in vitro.

DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

The delamination of pro-endocrine precursors after transit through the transient Ngn3HI commitment state might be a critical process for allowing the epithelium to go through more rounds of endocrine precursor production over an extended period. Without timely exit, this cellular flow would be blocked, and the epithelium could become stuffed with mixed undifferentiated and differentiated endocrine cells. The consequence could be massive feedback effects on the intercellular communication processes that give birth to endocrine-committed cells, and subsequent distorted allocations among the various hormone-secreting cell types, plus decreased islet cell mass. Here again, we see the importance of coordinating the cell biological process of morphogenesis with the endocrine cell ontogeny programs. It is also likely that a proper apico-basal polarization of the epithelium will be necessary to accommodate and direct the delamination process.

We can, therefore, expect to see future focus on how pro-endocrine cells escape the epithelium, using asymmetric cell division (ACD) or epithelial-to-mesenchymal transition (EMT), and if these processes overlap and are less than mutually exclusive. EMT-based escape involves 5 steps: (1) a post-mitotic cell breaking tight junction connections with its neighbors, (2) losing epithelial adherence (E-cadherin down-regulation), (3) transiently acquiring motility and other mesenchymal characteristics, (4) epithelial exit, and (5) the re-formation of tight junctions between remaining cells across the space vacated (Morales et al.,2007). Despite the release of pro-endocrine cells from the trunk epithelium being proposed to involve EMT, concrete evidence of the actual process or components involved has yet to be provided. A tantalizing suggestion comes from studies of Snail2, a well-known inducer of EMT and cell movement, which was found in scattered cells of the trunk epithelium during the secondary transition. As 80% of Ngn3HI cells are Snail2+, this suggests that endocrine precursors initiate EMT very soon after commitment to endocrine fate (Rukstalis and Habener,2007). A deeper analysis of Snail2 function in endocrine delamination and any “epithelial back-up effects” when it is blocked will be required.

In classical ACD, an orthogonal division plane does not split the apical membrane, and generates an apical daughter that retains tight-junction continuity with the epithelial sheet, and a basal daughter cell that does not (although skewed divisions can also produce the same outcome). Commonly in ACD, the apical cell retains progenitor-status, whereas the basal cell becomes postmitotic and begins differentiation; both daughters initially retain some epithelial character. This mechanism of progenitor cell self-renewal coupled with cell commitment-differentiation has been reported during neurogenesis in the developing central nervous system, and in dividing muscle satellite stem cells during regeneration (Huttner and Kosodo,2005; Kuang et al.,2007). Whether the delamination of pancreatic pro-endocrine cells mainly involves ACD, or a mixture of both ACD and EMT, requires further exploration. Defining the in vivo β-cell birth niche more completely is an extremely active area of research. The relationship between the cellular context of endocrine progenitors, intra-epithelial or delamination/migratory state, and timing of allocation of the specific hormone-expressing cell fate has yet to be established.

The endocrine cells that are initially formed after delaminating from the epithelium aggregate into long stretches of interconnected cell clusters in close association with the tubular epithelium (a sort of “beads on a string” appearance). Shortly before birth in the mouse (∼E18.5), islets are formed by a separation/fission process in which α-cells engulf the β-cell clusters, initiating the formation of the mature peripheral mantle/core structure. The forming islets migrate away from the ductal epithelium and acquire their characteristic ovoid shape shortly after birth (Miller et al.,2009). The islet morphogenesis process requires the function of Rac, a Rho-family small GTPase. Blocking Rac1 function in nascent β-cells impairs β-cell migration, and islet clusters remain much more closely in contact with the duct epithelium, possibly as a result of a failure of Rac1-deficient cells to reduce their E-cadherin level (Greiner et al.,2009).

Building the appropriate islet architecture would be expected to yield the cellular communication required for the proper physiological behavior of endocrine cells. Cell-cell contact within the core of β-cells, combined with sufficient vascularization, is critical for mature β-cell function. Glucose-stimulated insulin secretion (GSIS) was recently found to require bidirectional β-cell communication via ephrinA and EphA. EphA forward signaling inhibits insulin secretion, whereas ephrin-A reverse signaling stimulates insulin secretion. Under basal conditions, β-cells use EphA forward signaling to suppress insulin secretion but then, in response to glucose, they shift to ephrin-A reverse signaling to enhance insulin secretion (Konstantinova et al.,2007). Mutation of a number of extrinsic factors and TFs leads to disrupted islet organization and impaired islet function. An oft-encountered phenotype with such mutants is the mixed-islet morphology, with α-, δ-, and PP cells mixed into the islet β-cell core, rather than remaining at their normal mantle location. A few examples of mutants with defective islet architecture include: (1) the BMPR1a deletion mutant (Goulley et al.,2007); (2) mice with persistent expression of HNF6 within the pancreas (Gannon et al.,2000); (3) mutants carrying a repressor form of Nkx2.2 (Doyle et al.,2007); and (4) CTGF null mutants (Connective Tissue Growth Factor, not a growth factor per se, but a likely important multi-functional regulator of multiple signaling pathways [Wnt, BMP, TGFβ]; Crawford et al.,2009).

EXOCRINE CELL DEVELOPMENT

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Ducts and Centroacinar Cells

The mature ductal network has four subcompartments: (1) a main duct as the common conduit to the duodenum; (2) the interlobular ducts connected to the main duct; (3) intralobular ducts draining exocrine enzymes into the interlobular ducts; (4) intercalated ducts, also called terminal ducts or centroacinar cells (CAC), which connect the acini with intralobular ducts (Ashizawa et al.,2005; reviewed in Grapin-Botton,2005; Cleaver and MacDonald, 2010). We currently have superficial information, at best, on the functional heterogeneity of duct cells within each of these subpopulations, or if there are, for example, different plasticity states between or within these groupings that render them more or less susceptible to the cellular reprogramming discussed later in this review (see “Facultative Progenitors Activities and Reprogramming Towards β-cells” section). It is possible that exploration of these issues will include substantial work in zebrafish, if the various duct types can be marked effectively and followed for long enough in living fish. How the various types of duct emerge from the complicated plexus remodeling events could also be revealing. We refer the reader to reports such as that by Dong et al. (2007).

Extensively abnormal duct development was reported in both global and pancreas-specific HNF6 gene inactivation (Jacquemin et al.,2003; Pierreux et al.,2006; Zhang et al.,2009), although these duct defects are likely relatively directly connected to an earlier problem in the development and maturation of the secondary transition trunk epithelium, and not in the differentiation program of duct cells per se. One problem in dissecting these phenotypes effectively is centered on our current inability to define the effect as occurring in proper duct-committed and differentiating cells, rather than the cells in the “duct epithelial” tissue of the secondary transition trunk epithelium, which transiently represents a bipotent pool of endocrine and duct-competent progenitors. Because HNF6 is required for the proper behavior of the pancreatic progenitor pool, and particularly for the induction of Ngn3 expression, the failure of endocrine specification and commitment in HNF6 mutants, and absence of subsequent delamination, could cause mass default into ductal fates, such that the epithelial overstuffing produces secondary defects in epithelial architecture. Problems with forming the endocrine-competent progenitor pool within the maturing epithelial arbor fits with the finding of defects in HNF6 mutants as early as E15.5, and generation of a cystic multilayered “piled up” epithelium. The intercalated ducts, which we propose could normally become partitioned, relatively early on, from the main endocrine/duct-competent trunk epithelium as part of the program of architectural resolution of the epithelial plexus, can apparently still form and acini differentiate, but there are no second-wave endocrine cells (Jacquemin et al.,2000; Pierreux et al.,2006; Zhang et al.,2009). The dilated multilayered epithelial cells in the HNF6 null lack primary cilia but maintain polarity as marked by apical Mucin1 (Muc1). HNF6 has not been linked directly to cilia formation in other organs, but HNF1β is a well-known regulator of cilia formation in the kidney, where its loss causes polycystic kidney disease. Thus, the lack of primary cilia might be explained by loss of HNF1β in HNF6 mutants. The role of HNF1β in early trunk epithelium development, versus differentiation of mature duct cells proper, and if it is similar to HNF6, remains to be determined. It will also be necessary to understand the epistatic and inter-regulatory relationships between HNF6 and other duct-specific TFs, such as Sox9 (Seymour et al.,2007) and Hes1 (Fukuda et al.,2006) in maintaining proper duct progenitor identity and differentiation programs in the mature pancreas.

“Global deletion” of Pdx1 or Ptf1a in the early foregut epithelium blocks pancreas development and subsequently allows only large cystic ducts to form (Offield et al.,1996; Kawaguchi et al.,2002). Deleting Pdx1 function at progressively later stages using an on-off doxycycline-regulated transgenic/knock-in system allows formation of interlobular ducts first, followed by intralobular ducts (Hale et al.,2005). It is possible that the large cystic ducts formed in the Pdx1 and Ptf1 null mutants adopted a common bile duct or intestinal character, and thereby lost the competence and microenvironment to produce the proper numbers of second-wave endocrine cells, but this hypothesis remains to be tested. Since the main pancreatic duct shares many common characteristics and markers with the EHB system, as well as a close proximity, these two tissues may interconvert in the absence of essential organ-specific maintenance cues (intrinsic and extrinsic). Indeed, it has even been shown that endocrine clusters, including β-cells, occur naturally in the extrahepatic bile ducts of adult mice (Dutton et al.,2007). The plasticity of the early foregut endoderm is further demonstrated in global Hes1-deficient mice, where significant amounts of ectopic pancreatic tissue containing islets and acini were closely associated with the common bile duct (Sumazaki et al.,2004; Fukuda et al.,2006).

The CAC lie at the junction of the acinar and the adjacent ductal epithelium. Some reports even suggest that they represent a thin internal sheath of cells lining the inside surface of the acinar cap, such that acini do not actually contact the duct lumen. CAC express several common duct-specific TFs, such as Sox9 (Seymour et al.,2007), HNF1β (Solar et al.,2009), and Hes1 (Stanger et al., 2005; Kopinke et al.,2011). Prior findings suggest that, under injury conditions, CAC in the adult pancreas may act as facultative progenitors competent to produce all three pancreatic cell types (Leeson and Leeson,1986; Gasslander et al.,1992; Hayashi et al.,2003; Nagasao et al.,2003). The CAC could presumably, at a low level, feed progeny towards the normal homeostatic maintenance of the various pancreatic cell types. A study by Rovira et al. (2010) detected high relative expression of aldehyde dehydrogenase1 (ALDH1, mainly ALDH1a1 and ALDH1a7) specifically in E-cadherin+ CAC. Isolated ALDH1+/E-cadherin+ cells were reported to give rise to all pancreas cell types from small aggregates (“pancreatospheres”) in vitro, and when implanted into cultured pancreatic explants. This observation might provide the strongest support yet for the long-standing proposal that CAC can act as facultative progenitors capable of transdifferentiation (Rovira et al.,2010).

Acinar Formation

Acinar differentiation starts at the distal tip epithelium at around E13.5. At this stage, the tip MPC undergo a switch to the pro-acinar fate by down-regulating sets of progenitor-specific TFs that maintain MPC status, while upregulating expression of Ptf1a and various digestive enzymes (Seymour et al.,2007; Solar et al.,2009; Schaffer et al.,2010; Esni et al., 2004). The terminally differentiated acinar cell is a factory dedicated to synthesis, storage, and regulated release of approximately 24 digestive enzymes and accessory proteins. Mammalian acinar cells are packed with rough endoplasmic reticulum, have a huge and very active Golgi apparatus, an apical region dominated by zymogen granules, and a massive mitochondrial complement with prodigious energy potential (Padfield and Scheele,1993; van Nest et al.,1980).

In comparison to endocrine development, fewer transcription factors have been shown to be involved in acinar development. Ptf1a (as described earlier in this review) is the key player in acinar formation; its loss causes acinar agenesis. As described above, early PTF1RBP-J activates RBPJL expression, and switching to PTF1RBP-JL initiates the pro-acinar differentiation program during the secondary transition. PTF1RBP-JL binds and activates both Ptf1a and RBPJL expression, enforcing a strong positive regulatory loop to ensure continued production of Ptf1a and RBPJL in acini (Masui et al.,2010). The PTF1RBP-JL complex can bind promoters of all the genes encoding secretory zymogens, including amylase, elastases 1 and 2, CpaI, chymotrypsin B, trypsin, and 50 or so additional genes involved in terminal differentiation and mature function of acini (Masui et al.,2010).

Mist1, a bHLH factor, is required to establish proper apical-basal polarity and full acinar differentiation (Pin et al.,2001). Initiation of acinar development occurs in Mist1-deficient mice, suggesting action downstream of Ptf1a. However, acini in Mist1−/− mice do not acquire the typical rosette-like morphology, possibly as a consequence of loss of functional gap junctions that was attributed to reduced connexin-32 levels (Zhu et al.,2004). Mist1-null acini lose apical-basal polarity (Pin et al.,2001) and exocytosis is impaired with intracellularly misdirected zymogen granules (Rukstalis et al.,2003). Ultimately, the mature pancreas has acinar-to-ductal metaplasia and acinar autodigestion. Recently, Jia et al. (2008) suggested that Mist1 controls acinar proliferation by increasing the level of the negative cell cycle regulator p21CIP1/WAF1. Deleting Mist1 in mature acini causes increased proliferation via reduced p21CIP1/WAF1 expression. Thus, Mist1 plays a dual role in maintaining a stable acinar cell identity and blocking acinar proliferation.

To date, canonical Wnt signaling is the only reported extrinsic cue with direct implication on acinar development. Acinar differentiation fails early on in the absence of β-catenin (Murtaugh et al.,2005; Dessimoz et al., 2005; Wells et al.,2007). Conversely, pancreas-specific deletion of adenomatous polyposis coli function (APC, endogenous β-catenin inhibitor) (Strom et al.,2007) or β-catenin stabilization (Heiser et al.,2006) at the onset of acinar differentiation results in their massive proliferation, generating an exocrine pancreas twice the normal size. Wnt/β-catenin roles in acinar development may, in part, be mediated by a key downstream target, Myc, whose deletion also impairs acinar formation and proliferation (Nakhai et al., 2008b). Although early suppression of canonical Wnt signaling is required for proper expansion of pancreatic progenitor numbers, later activation of Wnt signaling in the acinar compartment is required for acinar formation and proliferation (Heiser et al.,2006). It would be interesting to examine the interaction and possible epistatic relationship between Wnt/β-catenin/Myc and Mist1 in controlling acinar cell proliferation.

HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

There is a paucity of information on the development of the human pancreas at the morphological and genetic level, and it seems obvious that a detailed understanding of human pancreas development could be crucial for learning the best methods of in vitro directed differentiation of hES/iPS cells towards functional β-cells. Furthermore, it is necessary to determine the specific differences in the genetic program between rodents and humans, so that in vitro differentiation is not misled by knowledge gained solely from rodent studies (D'Amour et al.,2006; Kroon et al., 2008). Despite ethical constraints on procurement of human fetal tissue, pioneering studies from Scharfman's group provide prodigious information on human pancreas development. These include: (1) early differentiation and proliferation of endocrine cell types (Polak et al.,2000); (2) ex vivo analysis of endocrine and exocrine differentiation in human fetal pancreas grafted under the kidney capsule (Castaing et al.,2001,2005); (3) establishment of in vitro culture system for human fetal pancreas (Ye et al., 2005); and (4) in vivo lineage tracing system for labeling β-cells (Scharfmann et al.,2008). In the next section, we will discuss the similarities and highlight distinctions between human and mouse pancreas development.

Human pancreas formation largely mimics the process in the mouse, and a relative timeline is provided in Figure 4A. Dorsal and ventral pancreas budding is first evident at 26–35 dpc (day post coitum), and fusion of the anlagen occurs at 6 weeks of gestation (G6w, gestation 6 weeks). The dorsal bud produces most of the head, body, and tail of the mature pancreas, whereas the ventral bud contributes to the inferior part of the head of the organ (Adda et al.,1984; Slack,1995; Polak et al.,2000; Piper et al.,2004).

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Figure 4. Human versus mouse pancreas development. A: Schematic time line comparing different stages of mouse and human pancreas development. The timing of endocrine cell appearance and acinar differentiation are noted. B: Schematic drawing showing different morphological phases of endocrine cell clustering and islet formation in human pancreas. E, embryonic day; G, gestational; w, week; GSIS, glucose stimulated insulin secretion.

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Although only limited studies have been performed, Pdx1, Ngn3, and Islet1 have been immunolocalized at a few stages in human fetal pancreas (Castaing et al.,2001, 2005; Piper et al., 2004; Jeon et al., 2009; Piper Hanley et al., 2010). The spatiotemporal patterns of these TFs suggest that similar transcriptional regulatory mechanisms operate in both human and mice. Pdx1 is found in pancreatic progenitors broadly through the epithelium from G7w (or perhaps earlier) onwards, and later in insulin-producing cells. It was found in occasional early, but not later, glucagon-producing cells (Jeon et al.,2009). Ngn3+ cells are found scattered in the epithelium at G9w, concomitant with the emergence of β-cells first at G8w, which are followed by α-cells and δ-cells at G9w (Polak et al.,2000; Sarkar et al.,2008; Jeon et al.,2009). A rare population (<5%) of insulin+/glucagon+ cells was also reported in the early human fetal pancreas. In contrast to the mouse primary transition, there seem to be few if any distinct first-wave endocrine cells found in human, and it therefore seems difficult currently to assume that there is a human equivalent of the mouse secondary transition, either in terms of morphology or any dramatic changes in endocrine-specific transcription regulator expression (Sarkar et al.,2008); there may be a single, extended transition. Nevertheless, the insitiation of exocrine differentiation at G11w could represent the beginning of the move to a human secondary transition, classifying on the basis of the emergence of the cell-specific differentiation programs.

There has been prolonged controversy over important functional differences between the human “mixed-islet” architecture and the canonical core (β-cell) and mantle (other endocrine) structure that is found in mice (see Fig. 4B). Islet-like clusters appear from approximately G11w in human pancreas. Unlike the mixed-islet architecture observed in human adult pancreas, the aggregated insulin and glucagon-expressing cells in the human fetal islets (around G14–16w) seem to be arranged similarly to the mouse adult islet (Jeon et al.,2009), with a β-cell core and peripheral mantle of α and δ-cells. However, at G19w, it is proposed that there is a transient separation (perhaps driven by migration and strong differential adhesion) of the peripheral α-cells and δ-cells, away from the β-cell core, to form juxtaposed homogeneously mono-hormone-producing clusters. Presumably, the mono-hormone islets reintegrate amongst each other after G22w, and there is significant intermixing to generate the adult islet architecture (Jeon et al.,2009) (Fig. 4B). How the core-mantle or intermingled architecture becomes linked to or is controlled by the vascular, neuronal, or other physiologically relevant input, and the degree to which there are species-specific variations in endocrine function according to the mixed or core/mantle structure, are areas needing further exploration.

Another potentially unique feature in the human pancreas concerns the higher proportion of endocrine cells that delaminate predominantly via cell-cluster delamination (leaving as aggregates) rather than by single-cell exit mechanisms. Earlier in this review, we covered the relevance of this process with respect to lateral inhibition models. It is intriguing to speculate on putative similarities between an endocrine-aggregate delamination process and that used in forming the α-cell clusters formed during the primary transition in the mouse (Ray MacDonald, UT Southwestern, Dallas, TX; personal communication). There are obvious implications for gaining control over the epithelial delamination and endocrine commitment strategies used in human pancreatic tissue, or in its replication from ESC/iPSC in vitro.

The rodent and human pancreas show significant differences in the proportions of the individual hormone-producing cell types, although as in mice any specific islet has its origin in multiple progenitors (Scharfmann et al.,2008). There are also comparative studies showing the differential distribution of endocrine cells amongst the various regions of the human and mouse pancreas. Greater numbers of PP cells are located in the human head region, with more α-cells and β-cells in the neck, body, and tail regions (Stefan et al.,1982; Brissova et al.,2005). Mouse islets comprise ∼75% β-cells, ∼20% α-cells, and ∼5% other endocrine cells; while human islets contain ∼50% β-cells, ∼40% α-cells, ∼10% δ-cells, and a few PP cells (Brissova et al.,2005). The importance of these differences is still unclear, and any relevance to endocrine cell function and glucose/energy metabolism requires further detailed analysis. Regarding the eventual building of islet organoids from ES or iPS cells in vitro for cell-based therapy, rather than pure populations of β-cells, we might need to generate a true human islet architecture, rather than try to replicate the one found in mouse.

FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

The inherent plasticity of the adult pancreatic organ is an area of great interest, accentuated when one fantasizes about controllably reprogramming a portion of, for example, the acinar cells towards the β-cell fate as a way of offsetting diabetes. Plasticity of a fully differentiated cell is considered here as the ability of a cell to change its epigenome and adopt a functionally distinct fate. While anecdotal evidence for plasticity has existed for many years, recent lineage tracing studies in mice provide direct evidence that the “irreversibly terminally differentiated” pancreatic cells are surprisingly plastic in certain contexts. Consequently, the burgeoning field of regenerative medicine has seen several model organism attempts to coax various differentiated cells towards β-cell fate in vivo. Several reports demonstrate that differentiated pancreatic cells can be reversed into a more progenitor-like state, or undergo end-state post-mitotic interconversion (also called transdifferentiation), under stimuli such as chemical or surgical injury, or specific genetic interventions. In this section, we will briefly discuss the reprogramming of various endodermal cell types towards β-cells under two conditions: (1) injury-induced reprogramming, and (2) genetic manipulation-based reprogramming.

Injury-Induced Reprogramming

The origin of β-cells formed in response to injury has been debated for over a century. However, recent advances in mouse lineage tracing technology seem to now suggest that even some of the most previously provocative, audacious proposals (particularly regarding transdifferentiation) may in fact be correct. Four main hypotheses were proposed for the origin of β-cells during the response to injury (see Fig. 5), including: (1) replication of pre-existing β-cells; (2) neogenesis from duct epithelium; (3) acini-to-endocrine or acini-to-duct-to-endocrine transdifferentiation; (4) endocrine cell interconversion, for example, α-to-β cells.

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Figure 5. Origin of β-cell during reprogramming paradigms. Four possible hypotheses proposed to explain the origin of β-cells under reprogramming models induced by injury (blue text) or genetic-based interference-type manipulations (red text). These include: (1) replication of preexisting β-cells; (2) neogenesis from duct epithelium; (3) acini-to-endocrine or acini-to-duct-toendocrine transdifferentiation; (4) endocrine cell interconversion (mainly α to β reprogramming). Ppx, partial pancreatectomy; CldU, 5-chloro-2-deoxyuridine (nucleotide analog); IdU, 5-iodo- 2deoyuridine; DTA, Diphtheria toxin A-based β-cell destruction; STZ, streptozotocin (β-cell destruction agent).

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Under normal physiological conditions, β-cell turnover is slow and decreases progressively with age (Teta et al.,2005,2007). Upon injury, it appears that at least some β-cells can re-enter the cell cycle. Evidence from genetic- and nucleotide analog incorporation-based lineage tracing studies, under partial pancreatectomy (Ppx) conditions, have demonstrated that duplication of pre-existing β-cells represents a predominant mechanism of generating new β-cells in normal pancreas and under injury response (Dor et al.,2004; Teta et al.,2007). The ability of β-cells to proliferate under such acute surgical injury is impaired in FoxM1 null mutants, leading to a dramatic decrease in β-cell mass (Ackermann-Misfeldt et al.,2008). However, these studies did not rule out the possible contribution of other sources to the β-cell compartment. (As an aside here, the quantitative methods used in most studies would likely miss even a substantial contribution from another source; the struggle is to identify the alternative source and use lineage-tracing-style pulse-chase labeling to define their ability to produce β-cells in the most robust manner.)

The suggestion that adult pancreatic ductal cells could give rise to insulin-producing cells upon injury, as a neogenetic birth process, can be considered similar to the embryonic phase of endocrine cell formation. It is interesting to consider whether a subset of the mature ducts (perhaps CAC) could return to an “embryonic epithelial cord state,” and thereby regain the ability to generate endocrine-biased progenitors and committed precursors. Pancreatic duct ligation (PDL) is an injury model that generates tissue damage and cellular responses similar to those in acute pancreatitis, and may elicit regenerative behavior from within the duct epithelium. A ligature around the main pancreatic duct results in distal damage, acinar involution, and an associated inflammatory response. Xu et al. (2008) demonstrated that Ngn3+ cells were induced in the duct epithelium after PDL, and thus identified a potential source of new endocrine cells in vivo. These Ngn3+ duct cells, upon isolation, could produce all endocrine cell types when cultured in Ngn3 null pancreatic bud explants in vitro. Although long term lineage-tracing to determine the origin of Ngn3+ cells was not conducted, their close proximity to the ducts is consistent with duct cells as the location of the facultative endocrine progenitors. From further studies, using independent PDL and β-cell destruction (combining alloxan and EGF/gastrin treatment) strategies, Solar et al. concluded that adult HNF1β-expressing mature duct cells could not produce β-cells following injury (Solar et al.2009). One caveat of this study, which is common to all experiments when inducible CreER is used, is that only 40–60% of the HNF1β-expressing ducts were labeled (via HNF1β-CreERT2), therefore it remains possible that subpopulations within the ducts, or a HNF1βLO duct population, were capable of yielding endocrine progeny.

Because of their vast numbers and developmental relationship to the pancreatic endocrine lineage, acinar cells could be an attractive source for reprogramming towards β-cells. For example, diverting just a few percent away for conversion to β-cells might not compromise overall organ function. Several in vitro acinar cell culture studies demonstrated a potential acinar to endocrine/duct conversion when treated with EGF, nicotinamide, or TGF-α (for example, Means et al.,2005; Minami et al.,2005). Upon caerulein treatment in vivo, acinar cells transiently dedifferentiate to a progenitor-like state, based on the observation of Pdx1, Foxa2, Sox9, and Hes1 positivity (Jensen et al.,2005). In this case, transient dedifferentiation of acinar cells is followed by acinar regeneration with no contribution of acinar lineage to ductal and endocrine lineages. The same conclusion was obtained from studies using Ppx in combination with acinar-specific lineage tracing (Desai et al.,2007). In the latter setting, Elastase-CreERT2-labeled acini were only capable of producing acinar cells, but not ductal and endocrine cells. Furthermore, via lineage tracing of acinar cells, with the same Elastase-CreERT2 in a model of hyperplastic duct formation, it was concluded that acini-to-duct transdifferentiation was a minor contributor, but the endocrine lineage was not analyzed (Strobel et al.2007). The same criticism can still be levied about missing possible cells of origin with low CreERT2-induced labeling efficiencies, which in that case were ∼30% of the total acinar pool. In principle, the full transdifferentiation potential of the acini and ducts might be revealed only when better, much higher efficiency labeling is achieved with new lineage-tracing tools. When TGF-α over-expression was used to generate duct hyperplasia, lineage-tracing of acinar cells using Elastase-Cre or villin-Cre suggested that most hyperplastic ducts were transdifferentiated from mature acini, and that any endocrine cells detected in/near these duct structures did not come from acini or hyperplastic duct cells (Blaine et al.,2010). Taken together, these studies have led to the same conclusion that acinar cells can only give rise to acinar and ductal cells, but not endocrine cells, upon injury-induced regeneration.

Endocrine α-to-β cell conversion represents another potentially bold angle for a regenerative medicine approach to curing diabetes. Thorel et al. (2010) selectively expressed the diphtheria toxin (DT) receptor and used DT administration to achieve near total β-cell destruction in mice. Cre-based lineage tracing was used to conclude that under ∼99% β-cell destruction, α-cells move through a bihormonal glucagon+/insulin+ state on their way to generate authentic insulin+ β-cells (Thorel et al.,2010). It is notable that such a deep lesion is needed, because α-to-β cell conversion was not seen under ∼95% destruction. Understanding the molecular mechanisms of α-to-β transdifferentiation in this model could lead to discovery of the endogenous signaling pathways or the epigenetic state that could then be engaged (perhaps even using simple pharmacological interventions) to coax controllable amounts of α-to-β transformation under normal physiological or in vitro culture conditions. It will certainly be important to understand why such reprogramming mechanisms are not engaged by the slightly lower amounts of β-cell loss. Furthermore, small molecule screening could also lead to rational therapeutic intervention employing α-to-β reprogramming in humans. An α-to-β transdifferentiation was also claimed in response to PDL together with β-cell destruction by alloxan treatment. However, genetic lineage-tracing was not carried out in this study to validate the authenticity of α-to-β conversion (Chung et al.2010).

Genetic Manipulation-Based Reprogramming

Reprogramming of adult cells via the forced expression of key developmental transcription factors has seen some important advances. Although it will likely be difficult to apply such methods in humans, we propose that such manipulations will inform as to the mechanisms of plasticity and the epigenomic alterations that need to be induced as facilitatory or directly instructive on cell fate. One landmark in reinvigorating such research comes from the finding that acinar cells could convert into β-cells in vivo when given an adenovirus-borne cocktail of three transcription factors (Ngn3, Pdx1, and MafA) (Zhou et al.,2008) (Fig. 5). The success of this mixture is likely explained by the respective TF activities: (1) Ngn3 aids acquisition of general endocrine competence; (2) Pdx1 helps establish a stable β-cell rather than other endocrine fates; (3) MafA augments Pdx1 function, but pushes the newly generated β-cell towards achieving the final mature, functional state (Gannon et al.,2008; Ahlgren et al.,1998; Gradwohl et al.,2000; Vanhoose et al.,2008). The reported induction of β-cells was sporadic, apparently occurring directly from the acinar state without replication or movement through a prolonged dedifferentiated state. The acini-derived β-cells did not cluster, perhaps because only few cells received all factors at appropriate relative doses, or because only certain acinar cells (as marked by Cpa1 expression) are competent to respond. These acini-derived β-cells could improve glucose homeostasis in diabetic mice, but the full spectrum of their physiological function was not analysed. Moreover, it is still unclear how duct, centroacinar cells, or other foregut intestinal tract tissues, sit with respect to resistance or open-ness to this kind of molecular reprogramming. At first sight, the transformation might seem miraculous, although the contribution of inflammation (which arises because of a virus-induced pancreatitis-like condition; Chen et al., 2004a) to this plasticity may be important and needs more study. Both acini and β-cells are dedicated to extremely high-level cargo packaging, storage, and secretion (of enzymes or hormone, respectively), and their lineal relatedness could mean that β-cells and acini contain some valuable fundamental epigenomic similarities. The reprogramming could, therefore, be rationalized as “simply” changing the upper level management in a very efficient factory.

Reprogramming has also been enforced via persistent expression of endocrine lineage allocation TFs, resulting in interconversion between closely linked cell types such as α-to-β or the converse β-to-α fate-switch. Persistent expression of Arx, the key determinant of α-cell specification, in the Pax6+ endocrine precursor population (Pax6-Cre;cArxOE; OE = overexpression) during embryonic stage, causes a dramatic reduction in β and δ-cells, concurrent with increased α- and PP-cells in adult pancreas. Furthermore, selective activation of Arx production in adult β-cells forces their transformation into α-cells (Collombat et al.,2007). The complementary conversion of α-to-β cells was reported in response to persistent Pax4 production in endocrine precursors (Pax6-Cre;Pax4OE) or mature α-cells (Glucagon-Cre;Pax4OE) (Fig. 5). In this latter situation, it was proposed that the extended neogenesis of α-cells from ducts, hypothesized as being induced by the lack of circulating glucagon, continues to pump conversion-ready cells (we hypothesize that they might not be mature, but more like pro-α-cells) into the islets, where their continual change into β-cells keeps the stimulus to produce yet more α-cells, a cycle that ultimately results in mega-islet formation. Even in the face of these remarkable giant islets, with their large insulin store, the mice develop diabetes; it is now being tested whether the hyperglycemia arises because of a severe acquired insulin resistance in the peripheral tissues (Collombat et al.,2009).

Overall, these lineage-switching and induced neogenesis reports start to generate significant excitement over the possibility of these processes being therapeutically manageable, and that we might one day be able to turn the process on and off as desired; for example, to induce the formation of appropriate numbers of α-derived β-cells by activating Pax4 for a limited period, then shutting it down before formation of mega-islets and development of secondary physiological problems. Related to this area of research, our own laboratory has begun to find evidence that the persistent expression of Pdx1 can effect a postnatal α-to-β cell conversion, without mega-islet formation and under maintained euglycemia (Y-P. Yang and C.V. Wright, unpublished data).

PERSPECTIVES

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Cell Biology Aspect of Pancreas Development

Despite the fact that the field of pancreas molecular developmental biology has existed for essentially only 15 years or so, its activity is progressively improving in rigor, becoming more systematic and process-oriented rather than focused on individual genes together with rather low resolution descriptions of normal and mutant cellular behaviors. A significant number of mutant phenotypes and reprogramming potentials related to TFs have been discovered, although we expect to see more focus in this respect on the possible signaling molecules and “niche environments.” We also hope to see these foundational discoveries being leveraged to a deeper analytical level. We envisage that we will, perhaps relatively soon, be able to unravel with precision all of the details related to cellular ontogeny, lineage diversification processes, and how they are mechanistically coupled to proliferation and organ size control.

A complementary area of focus on the cell biology of pancreas development should see a transition away from general tissue analysis to a precise dissection of single cell behaviors (such as has already been occurring in other organ systems), which will be facilitated by the rapid improvements in tools, reagents and molecular interrogation methods. A synthetic, and likely systems-based approach, could be necessary in order to gain a systematic view of how cells talk to each other during organogenesis, and how they interpret a multitude of external signals while also listening to their internal genetic instructions. The physiological function of the final cell types is the end-product of the combined entraining and intrinsic programs that are encountered throughout the cell's history. For the pancreatic endocrine cell, this developmental journey would start when its ancestors first land in the dorsal or ventral bud, to become modified by the various signals received as subsequent generations of cells decide whether they belong to the tip or trunk epithelial domain, how they then become endocrine-biased progenitors and then move forward and decide which hormone-producing cell fate they favor.

Continually being aware that any part of pancreas organogenesis represents an integration of intrinsic and extrinsic cues within a specific cellular context will help to produce rapid, major revisions of our views on lineage relationships and the stability of the final cell fates. Linking microlumen formation and the new complexities of epithelial morphogenesis to cell-fate specification is one useful new angle for the field, and the plexus remodeling process should provide a new framework to decorate with the precise location and behavior of the various progenitors and transitional intermediates. In this light, determination of the full complement of instructive intrinsic factor(s), as well as new molecular players that trigger microlumen formation and coordinate apical-basal polarization, could provide more direct information on the cell biological control of pancreas organogenesis, with special reference to how β-cells are born and mature. Taking these new discoveries as the stimulus and springboard, we will strive to see how niches and equivalence groups, competence windows, and plasticity resistance, are all orchestrated within a dynamic epithelium.

In the next 5–10 years, we envisage much more insight with respect to the influence of extracellular matrix molecules, neurons, and mesenchyme on epithelial morphogenesis, delamination, and lineage allocation. More studies focusing on how islet neurons affect endocrine cell maturation and physiological function are needed. Our preference is to emphasize both the macro- and micro-environment, including the 3-D architecture of the epithelium that are linked to endocrine specification, and also, how a carefully timed epithelial delamination sends cells off to cluster into the islets of Langerhans with an internal knowledge of their prospective hormone type and physiological role. Finding ways of more effectively monitoring cell specification and progression along the differentiation program, and through key transitional intermediates, especially in real-time by 3-D imaging methods on live tissue, should be a major expansion. Breaking the entire epithelial plexus and arbor down in a reductionist manner to more manageable units, and understanding how to cross-reference between tissue landmarks, boundaries, and prospectively, rigorously characterized “marker” cell types may seem obvious, but we propose that it is time to push much harder in these areas.

Detailed molecular analysis of trunk epithelium biology, and where endocrine cells form during the secondary transition, will help in learning what induces the production and maturation of authentic β-cells, and how the window for producing them is opened or closed developmentally, in terms of hormone cell type and quantity. Again, real-time, high-resolution studies with fluorescently tagged marker genes or proteins, including those recently reported by Stewart et al. (2009), should be useful.

NEW TOOLS AND FUTURE TECHNOLOGIES

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Advances in gene-targeting methods to generate conditional and inducible gene knockout strains, and on-off regulatable systems, have allowed characterization of spatiotemporal function of key TFs. More precise Cre-loxP lineage-tracing methods will add more layers to lineage and epistatic relationships, and tracing more selective cell populations identified through their co-expression of multiple marker genes should be another focus. The addition of other recombinase driver/reporter strains, such as Flpe/FRT, σc31 intergrase/attP-attB, or the recent Dre/Rox (Sauer and McDermott,2004; Anastassiadis et al.,2009) to the Cre/LoxP reporter systems could help in this regard. Transgenic approaches with cell-specific Cre reconstitution (assembling active Cre from “split-Cre”) represent alternative strategies, although improvement is needed to overcome the relatively low current efficiency of reconstitution (see, for example, Xu et al.,2007). A combination of inducible and dual-reporter lineage tracing could allow examination of progenitor heterogeneity over time in rapidly changing cell populations.

Another area demanding attention is the identification of collections of cell surface molecules as markers to identify specific competencies in cell populations both prospectively during pancreas development, and to allow flow cytometric sorting and interrogation by gene expression profiling and epigenomic analysis. We envisage a reiterative process of refining the map of the ontogenetic transitional states. This information will aid us making better in vitro differentiation protocols for ES and iPS cells. An important application will be to assess the maturity status of β-cells generated from in vitro directed differentiation, as compared to authentic human β-cells.

EPIGENOMICS

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Epigenomics has recently attracted enormous attention in many organs, including the pancreas. For example, histone deacetylase (HDAC) and DNA methyltransferases (Dnmts) are required for cell specification and lineage allocation at distinct stages of pancreas development, as well as β-cell regeneration both in mouse and zebrafish (Haumaitre et al.,2008; Noel et al.,2008). Furthermore, control of β-cell proliferation in young and aged mice has been linked to the Polycomb group protein, Bmi1, operating via transcriptional suppression (by H2A ubiquitylation) of the β-cell proliferation inhibitor, Ink4a/Arf (Dhawan et al.,2009).

Nonetheless, many epigenomics-related reports study global deletion of histone modifier functions in the pancreas, which might obscure cell-type-specific roles. It will be useful to determine the epigenomic hallmarks of progenitor and progeny during development, as well as for the adult cell types, including in normal and diabetic conditions, as shown recently by van Arensbergen et al. (2010). Such information could facilitate improvements in α-to-β cell reprogramming via manipulating key epigenomic transitions. Information on alterations of the epigenome in response to signaling factors during organogenesis could be used to improve ES cell movement towards β-cells and in ascertaining the trueness of the process.

ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Current in vitro directed differentiation protocols from human ES cells generate β-cells that are immature, although intermediate progenitor clusters, implanted and matured in mice, produce excellent quality β-cells (D'Amour et al.,2006; Kroon et al.,2008). Much is missing from the in vitro method, perhaps because of the incapacity to support β-cell maturation, or because β-cell-directed entraining is just slightly off-track, even from an early phase, with respect to normal endocrine ontogeny (see, for example, Yang and Wright,2009). In addition, hES cells are generally grown by relatively two-dimensional (2-D) monolayer methods, which might prevent the 3-D generation of the niches needed for proper β-cell birth and maturation. This latter point may be critical given the tissue complexities and multiple interactions that we have reviewed, and optimizing the 3-D culture system to include ECM components reminiscent of the developing pancreas should be considered. One could also find ways of tricking hESC grown in vitro into responding as if they were in the normal 3-D environment, and approaching novel biopolymers for support and differentiation (Kreger et al.,2010), even in combination with nanoscale bioengineering (von der Mark et al.,2010), could be excellent supplemental directions.

Small molecule chemical library screening might identify imitators of the cellular signaling events that normally guide pancreas formation, or represent a clean, simple method to relax the cellular resistance to plasticity in non-β-cells, both in vivo and in vitro. Maybe, for instance, we could find ways of making α-cells very efficiently in huge numbers, and screen for small molecules to convert them en masse into β-cells.

EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

Despite our focus for ethical and research tool reasons on rodent pancreas development and endocrine cell formation, a coherent understanding of human pancreas development is definitely most significant. While mouse and human pancreas development and function may be similar, we attempted to highlight some differences earlier in this review, and we foresee a fairly dramatic drift towards direct studies on human tissue. We might establish mature human β-cell primary cell lines, which could be dedifferentiated slightly, propagated in vitro, then redifferentiated to β-cells. We can likely gain access to viable human fetal or adult pancreatic tissue for α-to-β cell or acinar-to-β cell reprogramming studies in vitro. Xenotransplantation of fetal pancreas fragments into suitable mouse hosts might allow more detailed longitudinal studies of the endocrine developmental process.

Acknowledgements

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES

We thank Spencer Willet, Eric Bankaitis, Lindsay Marjoram and Guoqiang Gu for critical comments and valuable suggestions on the manuscript, and are grateful to Justin Wells for technical and artistic expertise in preparing the figures. This work was supported by the NIDDK grant number 1 UO1 DK089570-01.

REFERENCES

  1. Top of page
  2. Abstract
  3. OVERVIEW OF PANCREAS DEVELOPMENT
  4. PRE-PANCREATIC ENDODERM PATTERNING AND PANCREAS INDUCTION: INTERPLAY BETWEEN EXTRINSIC AND INTRINSIC FACTORS
  5. DORSAL PANCREAS INDUCTION: SIGNALING FROM NOTOCHORD AND VASCULAR TISSUE
  6. VENTRAL PANCREAS INDUCTION
  7. PANCREAS BUDDING AND THE PRIMARY TRANSITION
  8. EARLY PANCREAS TRANSCRIPTIONAL PROGRAM: PLASTICITY OF THE EARLY PANCREATIC BUD
  9. NOTCH SIGNALING IN EARLY PANCREATIC PROGENITOR DEVELOPMENT
  10. POLARIZATION AND TUBE FORMATION FROM THE PROTODIFFERENTIATED EPITHELIUM
  11. BRANCHING MECHANISMS AND PROGENITOR DOMAIN COMPARTMENTALIZATION
  12. “MESENCHYMAL- EPITHELIAL” CROSSTALK IN PANCREAS MORPHOGENESIS
  13. THE SECONDARY TRANSITION: ONSET OF ISLET, DUCT AND ACINAR DIFFERENTIATION
  14. THE TRUNK EPITHELIUM DURING THE SECONDARY TRANSITION
  15. ENDOCRINE SPECIFICATION
  16. ENDOCRINE SUBTYPE SELECTION, DIFFERENTIATION, AND MATURATION
  17. CLASS I: GENERAL ENDOCRINE PRECURSOR DIFFERENTIATION FACTORS
  18. CLASS II: LINEAGE ALLOCATION FACTORS
  19. CLASS III: MATURATION FACTORS
  20. DELAMINATION OF PRO-ENDOCRINE CELLS AND ISLETOGENESIS
  21. EXOCRINE CELL DEVELOPMENT
  22. HUMAN PANCREAS DEVELOPMENT: A COMPARISON TO MOUSE
  23. FACULTATIVE PROGENITOR ACTIVITY AND REPROGRAMMING TOWARDS β-CELLS
  24. PERSPECTIVES
  25. NEW TOOLS AND FUTURE TECHNOLOGIES
  26. EPIGENOMICS
  27. ES CELL DIFFERENTIATION SYSTEMS AND SMALL MOLECULE LIBRARY SCREENING
  28. EX VIVO/ IN VITRO HUMAN ISLET STUDIES: ALTERNATIVE β-CELL SOURCES
  29. Acknowledgements
  30. REFERENCES
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