Pancreas Organogenesis: From Lineage …msanderlab.org/wp-content/uploads/2017/07/Shih-2013...the cardiac mesoderm surrounding the prehepatic region (Deutsch et al. 2001, Rossi et

CB29CH04-Sander ARI 29 August 2013 16:20

Pancreas Organogenesis:From Lineage Determinationto MorphogenesisHung Ping Shih,∗ Allen Wang,∗ and Maike SanderDepartments of Pediatrics and Cellular & Molecular Medicine, Pediatric Diabetes ResearchCenter, University of California, San Diego, La Jolla, California 92093-0695;email: [email protected]

Annu. Rev. Cell Dev. Biol. 2013. 29:81–105

First published online as a Review in Advance onJuly 31, 2013

The Annual Review of Cell and DevelopmentalBiology is online at http://cellbio.annualreviews.org

This article’s doi:10.1146/annurev-cellbio-101512-122405

Copyright c© 2013 by Annual Reviews.All rights reserved

∗Equal contributions.

Keywords

pancreas, development, progenitor, exocrine, endocrine, pluripotent stemcell

Abstract

The pancreas is an essential organ for proper nutrient metabolism and hasboth endocrine and exocrine function. In the past two decades, knowledgeof how the pancreas develops during embryogenesis has significantly in-creased, largely from developmental studies in model organisms. Specifically,the molecular basis of pancreatic lineage decisions and cell differentiation iswell studied. Still not well understood are the mechanisms governing three-dimensional morphogenesis of the organ. Strategies to derive transplantableβ-cells in vitro for diabetes treatment have benefited from the accumu-lated knowledge of pancreas development. In this review, we provide anoverview of the current understanding of pancreatic lineage determinationand organogenesis, and we examine future implications of these findings fortreatment of diabetes mellitus through cell replacement.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82OVERVIEW OF PANCREAS DEVELOPMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82LINEAGE DECISIONS DURING PANCREAS DEVELOPMENT. . . . . . . . . . . . . . . 85

Pancreas Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85Establishing and Maintaining Pancreatic Identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87Signaling During Early Pancreatic Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88Compartmentalizing the Organ: Patterning of Tips and Trunk . . . . . . . . . . . . . . . . . . . 89Development of the Acinar Lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90The Endocrine Versus Ductal Cell Fate Choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Differentiation of the Islet Cell Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

PANCREAS MORPHOGENESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93Intracellular Signals Governing Pancreas Morphogenesis . . . . . . . . . . . . . . . . . . . . . . . . . 94Coordinating Morphogenesis and Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94Morphogenetic Changes and Endocrine Cell Differentiation. . . . . . . . . . . . . . . . . . . . . . 95

APPLICATION OF DEVELOPMENTAL PRINCIPLES TOWARD STEMCELL DIFFERENTIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95Generating Pancreatic Progenitors from Human Pluripotent Stem Cells . . . . . . . . . . 96Beyond Progenitors: How to Generate Functional Endocrine Cells In Vitro . . . . . . . 96Human Pluripotent Stem Cells as a Model for Understanding

Pancreas Biology and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97FUTURE PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

INTRODUCTION

The pancreas is essential for nutrient metabolism and has both exocrine and endocrine functions.The exocrine pancreas consists of acinar and ductal cells, whose prime role is nutrient diges-tion. The acinar cells secrete pancreatic juice containing enzymes that catalyze the breakdownof proteins, carbohydrates, and lipids. Upon release from the acinar cells, the pancreatic juice istransported into the duodenum via the ductal system (Figure 1). Functioning largely independentfrom exocrine cells, the endocrine pancreas is responsible for regulating glucose homeostasis byreleasing hormones into the blood stream. The endocrine pancreas comprises five endocrine celltypes, which reside in small cell clusters called islets of Langerhans.

The biology of the pancreas has been studied intensely, largely driven by the hope of findingbetter treatments for devastating pancreatic diseases, such as diabetes mellitus, pancreatitis, andpancreatic adenocarcinoma. In particular, advancements in stem cell technology have recentlysparked optimism that diabetes could be cured by harnessing stem cells for therapeutic use. This hasled to heightened interest in understanding embryonic development of the pancreas, specificallythe events involved in cell fate decisions and endocrine cell differentiation. Recent successes inderiving pancreatic cells from human pluripotent stem cells (hPSCs) prove exemplary for thefruitful application of developmental principles to stem cell differentiation.

OVERVIEW OF PANCREAS DEVELOPMENT

The invention of genetic tools, in particular transgenic mice, has been instrumental in defining themolecular events leading to pancreas organogenesis. Genetic lineage-tracing experiments revealed

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Liver

Gallbladder

SpleenSpleen

Pancreas

Stomach

Duodenum

Commonbile ductCommonbile duct

Endocrinecells

Acinar cells

PancreaticductsArtery

Vein

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Islet of Langerhans

Figure 1Overview of adult pancreas anatomy. The pancreas lies behind the stomach in the abdomen and attaches to the duodenum. Forillustration purposes, the stomach is outlined with a dashed line and the duodenum is shown as a cut view. Exocrine acinar cells secretepancreatic juice containing enzymes for the breakdown of proteins, carbohydrates, and lipids. The pancreatic juice is transportedthrough a ductal network that releases the juice into the duodenum. The endocrine pancreas consists of small cell clusters, called isletsof Langerhans, containing five endocrine cell types, namely glucagon-producing α-cells, insulin-producing β-cells,somatostatin-producing δ-cells, pancreatic polypeptide-producing PP cells, and ghrelin-producing ε-cells. Note, the figure shows theislet organization characteristic for mice with centrally located β-cells. Pancreatic islets are surrounded by a dense capillary networkthrough which the hormones are released into the blood stream. See inset for an enlarged cutaway view of the pancreas.

a common origin of pancreatic exocrine and endocrine cells from a pool of progenitor cells inthe gut endoderm (Gu et al. 2002). In mice, pancreas development first becomes morphologicallyevident around embryonic day (e) 9.00 with the emergence of epithelial buds on opposing sides ofthe foregut endoderm (Figure 2a). The two pancreatic buds subsequently elongate alongside thepresumptive duodenum and stomach and, as a result of gut rotation, eventually fuse into a singleorgan by e12.5 (Figure 2b). Simultaneously, cell proliferation leads to a rapid size increase of thepancreatic buds, which also change their shape and begin to form branched tubular structures in aprocess known as branching morphogenesis. During this early phase of pancreatic development,which has been referred to as the primary transition (Pictet et al. 1972), the still-undifferentiatedpancreatic epithelium is tightly surrounded by mesenchymal cells.

During the secondary transition from e12.5 until birth, the pancreatic epithelium continuesto expand and branch into a complex yet highly ordered tubular network (Figure 2c). Thesemorphologic changes are paralleled by the differentiation of endocrine, acinar, and ductal cells.By the end of the secondary transition, the mouse pancreas has largely acquired the topologicalorganization of the mature organ, with clusters of acinar cells coalesced around the ends of theductal network and endocrine cell aggregates scattered throughout the organ (Figure 2c,d ).

www.annualreviews.org • Pancreas Organogenesis 83

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Trunk cells

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Over the past two decades, significant progress has been made in deciphering key molecularmechanisms that underlie the multiple lineage decisions leading to the formation of the pancreaticcell types. In comparison, far less is known about the regulation of pancreas morphogenesis andwhether a connection exists between morphogenetic events and cell differentiation. This reviewcovers current knowledge of these subjects and discusses how these discoveries have begun toenable the development of cell-replacement strategies for diabetes.

LINEAGE DECISIONS DURING PANCREAS DEVELOPMENT

Pancreas Induction

Prior to outgrowth of the pancreatic buds, a ventral and dorsal prepancreatic region is specifiedin the gut endoderm around e8.5 (Slack 1995). Although ventral and dorsal pancreatic budsarise independently in distinct locations, the molecular programs governing their specification areremarkably similar. In contrast to most epithelial cells of the early gut endoderm, the expression ofsonic hedgehog (Shh) is selectively excluded from the presumptive pancreatic endoderm (Hebroket al. 1998, Kim & Melton 1998). Transgenic misexpression experiments in mice revealed thatthe exclusion of Shh from the prepancreatic region is necessary for the induction of pancreaticmarkers (Apelqvist et al. 1997).

Like Shh, retinoic acid (RA) also participates in patterning the gut endoderm. Absence of RAleads to anteriorization of the endoderm, resulting in complete pancreas agenesis in zebrafish andlack of the dorsal pancreas in Xenopus and mice (Chen et al. 2004, Martın et al. 2005, Molotkovet al. 2005, Stafford & Prince 2002). RA seems to induce the dorsal pancreatic program by aShh-independent mechanism, because loss of RA is not associated with ectopic Shh expression inthe prepancreatic region in mice (Martın et al. 2005).

The ventral foregut region gives rise to both ventral pancreas and liver, which develop in closeproximity to each other (for review, see Zorn & Wells 2009). Fibroblast growth factor (FGF) andbone morphogenetic protein (BMP) signaling are critical prohepatic cues, which are secreted bythe cardiac mesoderm surrounding the prehepatic region (Deutsch et al. 2001, Rossi et al. 2001).The discovery of signals necessary for normal pancreas and liver induction has found practicalapplications in directed differentiation protocols of hPSCs, which use RA and inhibitors of Shhand BMP to induce pancreatic identity in vitro (D’Amour et al. 2006, Nostro et al. 2011).

Dorsal bud emergence appears to also be regulated by extrinsic signals from endothelial cells.The dorsal and ventral pancreatic buds develop from regions of foregut endoderm that are indirect contact with the endothelium of the fusing dorsal aortae and vitelline veins, respectively

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2Illustrated overview of pancreatic organogenesis. (a) The dorsal and ventral pancreatic epithelium evaginates into the surroundingmesenchyme between embryonic day (e) 9.0 and e11.5 in mice. At this stage, the pancreatic epithelium is comprised of a multilayeredcore of unpolarized cells engulfed by a basement membrane. Scattered microlumens (light yellow) arise between epithelial cells. Bloodvessels surround but have not yet penetrated the epithelial buds. (b) At e12.5, the outer tip cell layer ( green) of the pancreatic epitheliumforms recognizable branch protrusions. In the trunk portion of the pancreatic epithelium, microlumens fuse to form a primitive plexus,and groups of newly polarized cells ( purple) organize into rosettes around a lumen. At the same time, blood vessels begin to intercalateinto the epithelium and contact trunk cells. (c) At e15.5, the luminal plexus progressively remodels into a single-layered epitheliumconsisting of highly branched primitive ducts (also known as progenitor cords) and newly differentiated acinar cells. Ngn3-expressingendocrine precursors (orange) delaminate and migrate away from the progenitor cords to form endocrine clusters. Blood vessels areintercalated between nascent branches of the pancreatic ductal tree. (d ) In the mature pancreas, acinar cells cap the endings of smallterminal ducts and form functional exocrine secretory units. Endocrine cells are clustered in so-called islets of Langerhans, which arepenetrated by a dense network of blood vessels. See insets for enlarged views of depicted areas.


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(Lammert et al. 2001). Tissue recombination experiments and studies of endothelium-deficientFlk1−/− mice have shown that the absence of endothelial cells prevents induction of early pancreaticgenes (Lammert et al. 2001, Yoshitomi & Zaret 2004). However, contrary to these reports in mice,studies in zebrafish revealed pancreas specification in the absence of vascular endothelium (Fieldet al. 2003), a discrepancy that may reflect species differences.

Pancreaticprogenitors

Trunk

Nkx6.1

Tip

Ptf1a

Notch

Notch

Sox9Ngn3

Hes1

Oc1

Tcf2

Hes1

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Sox9 Gata4/6

Foxa1/2

Prox1

Oc1Tcf2

Glis3Prox1Sox9

Hes1

Acinus Duct

PTF1-L Nr5a2

Ptf1a

c-Myc

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Primarytransition

Secondarytransition

a

b

c

d

Oc1

Tcf2

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Nkx6.1

Nkx6.2

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α β

Arx Pax4 Nkx6.1 Pdx1

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Establishing and Maintaining Pancreatic Identity

Once the ventral and dorsal prepancreatic domains are specified, several transcription factorsbecome specifically expressed in the prospective pancreatic regions. Among the earliest transcrip-tion factors that mark this region are Pdx1, Ptf1a, and Sox9 (Ahlgren et al. 1996, Guz et al. 1995,Kawaguchi et al. 2002, Krapp et al. 1998, Seymour et al. 2007). Gata4/6, Foxa1/2, Tcf2, Onecut-1/2, Hes1, Prox1, and Mnx1 are also expressed in the prepancreatic domain, but their expressionexpands more broadly throughout the foregut endoderm (Figure 3a) (comprehensively reviewedin Gittes 2009, Pan & Wright 2011, Seymour & Sander 2011).

Mice lacking any one of these factors display varying degrees of pancreas hypoplasia or agenesis;however, they still show pancreatic budding, which indicates that absence of one single factor doesnot entirely prevent initiation of the pancreatic program (reviewed in Gittes 2009, Mastracci &Sussel 2012, Pan & Wright 2011, Seymour & Sander 2011). Although budding of the pancreasis still observed, evidence strongly indicates that these transcription factors are important fordetermining pancreatic fate. For example, Ptf1a deletion leads to a reallocation of subsets ofpancreas-fated progenitors to other endodermal lineages. This was shown by in vivo lineage-tracing experiments, which revealed a contribution of Ptf1a-deficient cells to adjacent duodenaland common bile duct endoderm (Burlison et al. 2008, Kawaguchi et al. 2002). Further evidencethat these early pancreatic transcription factors possess pancreatic fate-determining activity comesfrom gain-of-function experiments in Xenopus, which have shown that constitutively active formsof either Pdx1 or Ptf1a can allocate liver progenitors to the pancreatic lineage (Horb et al. 2003,Jarikji et al. 2007). Also, when ectopically expressed, Pdx1 and Ptf1a in combination are sufficientto convert duodenal precursors to the pancreatic fate (Afelik et al. 2006). Thus, in a developmentalcontext, Pdx1 and Ptf1a can confer pancreatic identity to nonpancreatic cells. The extent to whichPdx1 and Ptf1a are also capable of activating pancreatic genes in nonpancreatic adult cells remainsto be investigated. A better understanding of context-dependent effects of these early pancreatictranscription factors on gene expression could lead to novel strategies for reprogramming fibro-blasts directly into pancreatic cells, as shown recently for neurons and cardiomyocytes (Qian et al.2012, Thier et al. 2012).

In addition to their role in pancreas specification, all early pancreatic transcription factors arenecessary for further development of pancreatic buds (for reviews, see Gittes 2009, Pan & Wright2011, and Seymour & Sander 2011). Mechanistically, the profound effect on the developmentof the organ caused by loss of only one single transcription factor may result from these earlyfactors functioning in a transcriptional network with extensive cross-regulation between individualfactors. For example, the expression of both Tcf2 and Ptf1a depends on Onecut-1 (Haumaitreet al. 2005, Maestro et al. 2003); simultaneously, Onecut-1 expression depends on Tcf2 and Ptf1a

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Lineage decisions during pancreas development. (a) During the primary transition, the transcription factors Pdx1, Prox1, Onecut-1(Oc1), Ptf1a, Foxa1/2, Tcf2, Sox9, Gata4/6, and Hes1 mediate expansion of pancreatic progenitor cells and maintain pancreaticidentity. (b) At the onset of the secondary transition, pancreatic progenitors adopt either tip or trunk identity. Notch activity promotesthe trunk while repressing tip identity. Cross-repression between Ptf1a and Nkx6.1 mediates tip/trunk separation. (c) Tip cells adopt anacinar phenotype, which is maintained by a positive-regulatory loop between PTF1-L and Nr5a2. Trunk cells are bipotential for theductal and endocrine cell fate. The ductal versus endocrine fate decision is controlled by graded Notch activity. High Notch promotesthe ductal fate by activating both Hes1, a repressor of Ngn3, and Sox9, an Ngn3 activator. At low Notch activity, only Sox9 is activated,leading to endocrine differentiation. (d ) Endocrine precursors further differentiate into five different cell types, of which only α- andβ-cells are depicted. Cross-repression between the α-cell determinant Arx and β-cell determinants Pax4, Nkx6.1, and Pdx1 separatesthe two lineages.


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(Poll et al. 2006, Thompson et al. 2012). Similar cross-regulation has been demonstrated betweenSox9, Pdx1, Hes1, and Ptf1a (Ahnfelt-Rønne et al. 2012; Hale et al. 2005; Seymour et al. 2007,2012; Thompson et al. 2012). Thus, elimination of a single factor has repercussions that ultimatelyaffect multiple components of the pancreatic program. The advent of genome-scale tools and theavailability of stem cell–based in vitro models of pancreas development (D’Amour et al. 2006,Kroon et al. 2008) now enable experiments to globally define the relevant transcriptional networks.Such knowledge should help inform reprogramming strategies for deriving pancreatic cells fromother somatic cells.

Signaling During Early Pancreatic Growth

Whereas we have a relatively detailed understanding of the transcription factors regulating earlypancreatic development, less is known about the signals controlling their expression. Both Hes1and Sox9 are regulated by Notch signaling (Kageyama et al. 2007, Shih et al. 2012), which suggeststhat Notch signaling is important for early pancreas growth. As evidence to support this, embryosdeficient for the Notch effector RBP-jκ have been shown to exhibit early arrest in pancreaticgrowth, similar in degree to that seen after Hes1 or Sox9 deletion (Apelqvist et al. 1999, Fujikuraet al. 2006, Jensen et al. 2000, Seymour et al. 2007). Hes1 deletion is also associated with upregu-lation of the cell cycle inhibitor p57 in pancreatic progenitors (Georgia et al. 2006). Thus, thereappears to be a direct link between Notch activity, transcription factor expression, and cell cycleregulation in the early pancreas. Known and putative roles for Notch in the developing pancreashave been expertly reviewed (Afelik & Jensen 2012).

In addition to Notch signaling within the epithelium, pancreatic growth is also controlled byextrinsic signals from the surrounding mesenchyme. Seminal studies in the 1960s demonstratedthe importance of the mesenchyme for early pancreatic growth (Golosow & Grobstein 1962,Rutter et al. 1964, Wessells & Cohen 1966). However, we have begun only recently to understandhow mesenchymal signals control growth of the early pancreas. The best-understood pathwaythat relays pro-proliferative signals from the mesenchyme to the epithelium is the FGF signalingpathway. During the primary transition, FGF10 is highly expressed in pancreatic mesenchyme,whereas its receptor FGFR2 is expressed throughout the epithelium (Bhushan et al. 2001,Dichmann et al. 2003, Seymour et al. 2012). Gain- and loss-of-function experiments in micefurther demonstrated that FGF10 stimulates progenitor cell proliferation and is required forearly growth of the pancreatic buds (Bhushan et al. 2001). FGF10 has been shown to regulatePtf1a and Sox9 expression in the epithelium ( Jacquemin et al. 2006, Seymour et al. 2012),which explains why FGF10 deletion results in a phenotype similar to that observed in eitherPtf1a- or Sox9-deficient pancreas (Bhushan et al. 2001, Kawaguchi et al. 2002, Seymour et al.2007). During the early growth phase of the pancreatic buds, Sox9, FGFR2, and FGF10 form afeed-forward loop, with mesenchymal FGF10 maintaining epithelial Sox9 expression and Sox9upholding receptivity to FGF10 by cell-autonomously controlling FGFR2 expression (Seymouret al. 2012). If this loop is disrupted at any point, pancreatic epithelial cells can no longer maintaintheir identity, and liver genes are activated instead (Seymour et al. 2012). This demonstratesgenetic coupling of early pancreatic growth and maintenance of organ identity.

Additional signaling pathways critical to epithelial growth include the EGF, Wnt, and BMPpathways (Ahnfelt-Rønne et al. 2010, Miettinen et al. 2000, Murtaugh et al. 2005). Wnt and BMPsignals appear not to be relayed to the epithelium; instead, they are transduced by mesenchymalcells (Ahnfelt-Rønne et al. 2010, Jonckheere et al. 2008, Landsman et al. 2011). It is unknownwhether the ligand is secreted by the epithelium or the mesenchyme itself. Thus, signaling be-tween the mesenchyme and epithelium likely involves multiple signaling loops. Recent in vitro


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studies by Sneddon et al. (2012) on pancreatic lineage intermediates derived from hPSCs supportthis contention, demonstrating that coculture with mesenchyme effectively expands progenitorsand that this effect is specific both to the mesenchymal cell line used and to the progenitor beingamplified. They further showed that no single growth factor or combination of factors is sufficientto reproduce the magnitude of effect of coculture with mesenchyme, reinforcing the notion thatmultiple factors cooperate to stimulate epithelial expansion. The identification of factors orches-trating pancreatic growth should be a high priority to aid the development of scalable protocolsfor the in vitro production of pancreatic cells.

Compartmentalizing the Organ: Patterning of Tips and Trunk

Shortly after the primary transition, the nascent pancreatic buds have not yet acquired the macro-scopic structure of the mature organ, nor have they formed the differentiated cell types necessaryfor pancreatic function. Instead, they are almost entirely composed of multipotent progenitor cells(MPCs). The transition of noncommitted MPCs into an organ with different cell compartmentsis a multistep process, of which the first step is the segregation of a tip and trunk domain at theonset of the secondary transition (Figure 3b) (Zhou et al. 2007).

During this time, the developing organ continues to grow rapidly. Concomitantly, the pancre-atic epithelium undergoes dynamic structural changes, resulting in multiple protrusions that budfrom the edges. These structures, marked by expression of Ptf1a, c-Myc, and Cpa, represent thetip domain; conversely, the inner cells representing the trunk domain are identified by Nkx6.1/6.2,Sox9, Tcf2, Onecut-1, Prox1, and Hes1 (Figure 3b) ( Jacquemin et al. 2003, Klinck et al. 2011,Kopinke et al. 2011, Kopp et al. 2011, Schaffer et al. 2010, Solar et al. 2009, Vanhorenbeeck et al.2007, Wang et al. 2005, Zhou et al. 2007). These morphological changes mark the beginningof a complex sequence of cell rearrangements (Villasenor et al. 2010), resulting in the matureorgan structure (discussed in detail in the section on Pancreas Morphogenesis). In addition to thechanges in organ shape, the formerly multipotent MPCs have taken the first step toward lineagecommitment. Lineage-tracing experiments revealed that the trunk predominantly gives rise tothe endocrine and ductal cell lineages, whereas the tips are quickly restricted to an acinar fate(Figure 3b) (Kopinke et al. 2011, Kopp et al. 2011, Pan et al. 2013, Solar et al. 2009, Zhou et al.2007).

The transcription factors Nkx6.1/6.2 and Ptf1a act as master regulators during this process.These factors are initially coexpressed in MPCs, but their expression domains entirely segregateduring tip and trunk compartmentalization: Nkx6.1/6.2 promote trunk identity and segregateto trunk cells, and Ptf1a has an equivalent role in the tip compartment (Schaffer et al. 2010).Mechanistically, this process is initiated by transcriptional cross-repression between Nkx6.1/6.2and Ptf1a (Schaffer et al. 2010). The example of Ptf1a, which first specifies pancreatic fate andlater governs acinar cell development (Kawaguchi et al. 2002, Krapp et al. 1998, Masui et al. 2010),illustrates that developmental transcription factors can regulate different processes depending oncellular context.

Genetic studies have provided insight into the signaling pathways regulating tip/trunk segrega-tion, revealing that Notch signaling plays an essential role in promoting trunk identity. Repressionof Notch signaling causes excessive tip formation at the expense of the trunk population (Afeliket al. 2012, Horn et al. 2012, Magenheim et al. 2011b). Conversely, constitutively active Notchprevents tip formation and promotes expansion of the trunk domain partly by activating the trunkmarker Nkx6.1 (Afelik et al. 2012, Esni et al. 2004, Murtaugh et al. 2003, Schaffer et al. 2010).Although Notch signaling clearly plays a pivotal role in coordinating tip and trunk segregation, itremains unknown how Notch is regulated during this process.


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Development of the Acinar Lineage

In addition to their role in digestion, pancreatic acinar cells are an attractive target for β-cell-replacement therapy, because they are plentiful and retain the cellular plasticity to undergoreprogramming into β-cells (Zhou et al. 2008). Moreover, because they are the initiating celltype for precursor lesions of pancreatic cancer (Kopp et al. 2012), acinar cells are also a studyfocus for oncologists. As discussed, acinar cells predominantly arise from precursor cells in thetip domain (Pan et al. 2013, Zhou et al. 2007). By e15.5, most tip cells have undergone acinarcell differentiation, and subsequent expansion of the acinar domain is largely driven by cellproliferation (Pan et al. 2013, Zhou et al. 2007).

Acinar cell differentiation is synergistically orchestrated by the transcription factors Ptf1a,Rbp-jl, and Nr5a2/LRH-1, which exhibit first tip cell–specific and later acinar cell–specific ex-pression (Holmstrom et al. 2011; Krapp et al. 1996, 1998; Masui et al. 2007, 2010; Rose et al.2001; Zhou et al. 2007). Prior to acinar cell differentiation, Ptf1a interacts with the b-HLH tran-scription factor Rbp-jκ (Beres et al. 2006, Masui et al. 2007, Miyatsuka et al. 2007). Together,these factors are responsible for the activation of Rbp-jl, a constitutively active, acinar-restrictedparalog of Rbp-jκ (Masui et al. 2007). During the secondary transition, Rbp-jl then replaces Rbp-jκ in the complex with Ptf1a. This complex, termed PTF1-L, establishes the acinar phenotype bydirect activation of essential acinar genes (i.e., genes encoding digestive enzymes and proteins forexocytosis of zymogen granules), as well as Ptf1a and Rbp-jl themselves (Masui et al. 2007, 2008,2010; Rose et al. 2001). Thus, PTF1-L induces and maintains the acinar phenotype through anautoactivation loop that sustains high expression of acinar genes (Figure 3c).

Similarly, Nr5a2, a direct target gene of Ptf1a, is necessary for acinar cell differentiation(Holmstrom et al. 2011, Thompson et al. 2012). Nr5a2 binds to and regulates a set of genessimilar to those regulated by the PTF1-L complex, and Nr5a2 and PTF1-L cooperate in theregulation of acinar cell–specific genes (Holmstrom et al. 2011). Together, these studies demon-strate that Ptf1a promotes acinar cell differentiation by initiating and stabilizing a gene-regulatorynetwork that drives acinar gene expression.

Although Ptf1a is an indispensable coordinator of acinar cell development, forced expres-sion of Ptf1a alone is not sufficient to effectively induce acinar cell differentiation (Schafferet al. 2010). Initiation of acinar differentiation may require other transcription factors function-ing in parallel with Ptf1a. One candidate is Mist1, which is strongly expressed in acinar cellsbut does not depend on the PTF1-L complex for its expression (Masui et al. 2010, Yoshidaet al. 2001). Mist1-deficient mice exhibit loss of differentiated characteristics of acinar cells, in-cluding loss of cellular polarity and defective exocytosis (Pin et al. 2001). Thus, the PTF1-Lcomplex, Nr5a2, and Mist1 are all required to maintain the acinar phenotype. Genome-wideanalysis of Mist target genes may reveal whether Mist1 occupies genomic regions similar tothose of PTF1-L and Nr5a2 and could therefore regulate acinar development jointly with thesefactors.

Less understood than acinar cell specification and differentiation are the mechanisms under-lying the rapid proliferative expansion of acinar cells until birth. Unlike Mist1 and Ptf1a, whichnegatively regulate acinar cell expansion ( Jia et al. 2008, Rodolosse et al. 2004), Nr5a2 promotesproliferation of exocrine cell lines (Benod et al. 2011, Botrugno et al. 2004), revealing Nr5a2 asa potential candidate for driving acinar cell expansion. Additional candidates include c-Myc andβ-catenin, whose inactivation in early pancreatic progenitors causes acinar cell hypoplasia (Bonalet al. 2009, Murtaugh et al. 2005, Nakhai et al. 2008, Wells et al. 2007). Temporally and spatiallycontrolled inactivation of other acinar factors in mice may yield additional insight into molecularcontrol of acinar cell differentiation, expansion, and maintenance.


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The Endocrine Versus Ductal Cell Fate Choice

Whereas tip cells are fated to become acinar cells, the trunk domain is bipotential and producesductal and endocrine cells (Figure 3c) (Kopinke et al. 2011, Kopp et al. 2011, Solar et al. 2009).After the tip and trunk domains have separated at around e12.5, cells in the trunk undergo extensivemorphogenetic changes to form a 3-D network of tubules lined by a single layer of polarizedepithelial cells (Hick et al. 2009, Kesavan et al. 2009, Villasenor et al. 2010). This network oftubules is referred to as the primitive ducts or progenitor cords and is the epithelium that givesrise to pancreatic endocrine cells (Hick et al. 2009, Kopp et al. 2011). During the secondarytransition, a subset of cells in the progenitor cords initiate expression of the transcription factorNeurogenin3 (Ngn3), which marks the onset of endocrine cell differentiation (Gradwohl et al.2000, Gu et al. 2002, Johansson et al. 2007, Schwitzgebel et al. 2000). Trunk epithelial cells that donot activate Ngn3 eventually contribute to the ductal tree (Beucher et al. 2012, Magenheim et al.2011b, Wang et al. 2010). Thus, precise control of Ngn3 induction is the key factor balancing theendocrine versus ductal cell fate decision.

Many of the same transcription factors (e.g., Sox9, Tcf2, Onecut-1, Hes1, Prox1, and Glis3)that segregate to the trunk domain during tip/trunk separation later become restricted to the ductsand excluded from the endocrine compartment (Figure 3c) (Delous et al. 2012, Kang et al. 2009,Maestro et al. 2003, Pierreux et al. 2006, Shih et al. 2012, Wang et al. 2005, Westmoreland et al.2012, Zhang et al. 2009). Consistent with their duct-specific expression, their inactivation in micecauses defects in ductal cell morphology and abnormal architecture of the ductal tree. For example,pancreata deficient for Sox9, Glis3, or Onecut-1 exhibit ductal cysts and lack ductal primary cilia,a functionally important organelle of ductal epithelial cells (Kang et al. 2009, Pierreux et al. 2006,Shih et al. 2012). Whether these ductal malformations reflect a functional requirement of thesefactors in the maintenance of ductal epithelial properties or a role in ductal cell differentiation isunknown. Conditional inactivation of the genes encoding ductal factors in developing and matureducts should further define their role in ductal development and function. This knowledge couldbe relevant to understanding the pathogenesis of duct-related illnesses, including pancreatitis andpancreatic ductal adenoma carcinoma.

Unlike ductal cell development, endocrine differentiation has been studied intensely, drivenby hopes that such knowledge could help produce replacement β-cells for diabetes therapy. Be-cause activation of Ngn3 expression is essential for initiating endocrine cell differentiation, therehave been substantial efforts to understand Ngn3 regulation. Interestingly, many transcriptionfactors controlling ductal development, such as Sox9, Tcf2, Glis3, and Onecut-1, are also nec-essary for Ngn3 induction ( Jacquemin et al. 2000, Kang et al. 2009, Kim et al. 2012, Lynnet al. 2007, Maestro et al. 2003, Seymour et al. 2008, Zhang et al. 2009), which indicates dualroles for these factors in the development of both lineages. However, Ngn3 is expressed in onlya subset of cells within the primitive ducts, which raises the question of how its expression isrepressed in the majority of embryonic ductal cells. Biochemical and genetic evidence suggeststhat the transcription factor Hes1 plays an important role in repressing Ngn3 transcription andpreventing widespread Ngn3 activation (Ahnfelt-Rønne et al. 2012, Apelqvist et al. 1999, Jensenet al. 2000, Lee et al. 2001). Recently, Hes1 was found to accelerate Ngn3 protein degrada-tion (Qu et al. 2013), which suggests that additional posttranscriptional mechanisms contributeto Ngn3 regulation. Supporting this idea, Villasenor et al. (2008) reported that Ngn3 mRNAis detected in a much broader domain than is the protein. Generally, the detailed mechanismscontrolling Ngn3 expression are still poorly understood. Progress has been slow owing to thelack of a cell system in which Ngn3 regulation can be studied ex vivo. Because Ngn3 activa-tion follows a pattern during pancreatic differentiation of hPSCs similar to that seen in vivo


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(D’Amour et al. 2006, Xie et al. 2013), hPSCs could prove an informative model for furtherstudies.

Because ducts simultaneously express positive and negative regulators of Ngn3, their relativelevels may determine whether or not a progenitor activates Ngn3 and adopts endocrine identity.Evidence suggests that the Notch pathway plays an important role in this decision. Constitutiveactivation of Notch signaling prevents Ngn3 activation and promotes ductal cell differentiation(Greenwood et al. 2007, Murtaugh et al. 2003). This suggests that bipotential ductal/endocrineprogenitors must escape Notch signaling to initiate endocrine differentiation. Surprisingly, Notchhas been found to induce both negative and positive regulators of Ngn3. High Notch signalingpromotes the expression of both Hes1 and Sox9, resulting in Ngn3 repression; conversely, lowerNotch activity promotes the expression of Sox9 alone, allowing for Ngn3 activation (Figure 3c)(Shih et al. 2012). Thus, the select activation of Ngn3 in small subsets of progenitors appears tobe controlled by graded Notch activity in the progenitor epithelium. How individual cells withinthe progenitor cords acquire different levels of Notch activity remains unknown.

After the secondary transition, endocrine precursors gradually stop arising from the ductal ep-ithelium, which suggests either that postnatal ducts lose competency to form endocrine precursorsor that proendocrine signals are missing (Kopinke et al. 2011, Kopp et al. 2011, Solar et al. 2009).Studies of pancreatic injury models and clonal analysis of ductal cells in vitro indicate that adultducts retain the capacity to activate Ngn3 and thus are competent to initiate, at least to some extent,endocrine programs ( Jin et al. 2013, Kopp et al. 2011). Adult ductal cell plasticity is a field of intensestudy that has been reviewed comprehensively (Desgraz et al. 2011, Reichert & Rustgi 2011).

Differentiation of the Islet Cell Types

Once Ngn3 is expressed, progenitors exit the cell cycle and delaminate from the progenitorcords into the surrounding stromal tissue (Gouzi et al. 2011, Miyatsuka et al. 2011). Althoughindividual endocrine precursors are unipotent (Desgraz & Herrera 2009), Ngn3+ cells as a wholegenerate five different endocrine cell types: glucagon-producing α-cells, insulin-producing β-cells, somatostatin-producing δ-cells, pancreatic polypeptide–producing PP-cells, and ghrelin-producing ε-cells.

Johansson et al. (2007) have shown that the competence of endocrine precursors to producedifferent endocrine cell types is temporally controlled. When Ngn3 expression was induced atdifferent developmental time points from a conditional Ngn3 transgene, progenitors first differ-entiated into α-cells, then β- and δ-cells, and finally PP-cells. The mechanisms controlling thischange in developmental potential are unknown. Studies in the developing neurons, muscle, andpituitary suggest that Notch signaling could be involved (Mizutani & Saito 2005, Mourikis et al.2012, Zhu et al. 2006). Here, the duration of exposure to Notch is critical for determining pro-genitor cell competence. If the pancreas uses a similar mechanism, optimizing Notch exposuretime could be key to maximizing β-cell yield in directed differentiation protocols for hPSCs.

When differentiating into hormone-producing cells, Ngn3+ endocrine precursors undergodynamic changes in gene expression, resulting in activation of both Ngn3-dependent transcrip-tion factors (Pax4, Arx, Rfx6, NeuroD1, Pax6, Isl1, and IA2) and hormones. Analysis of mousemutants revealed that these factors control many aspects of endocrine development, includingcell differentiation, cell maintenance, and islet cell–type identity (comprehensively reviewed inMastracci & Sussel 2012, Pan & Wright 2011). Here we focus on the mechanisms governing isletcell–type identity, specifically on the cell fate choice between the β- and α-cell lineage.

As with the mechanisms governing tip/trunk segregation, fate distinction between β- and α-cell precursors is thought to rely on mutual repression between opposing lineage determinants.


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The transcription factors Pax4, Pdx1, and Nkx6.1 act as critical β-cell determinants (Collombatet al. 2003, Gannon et al. 2008, Henseleit et al. 2005, Holland et al. 2002, Sosa-Pineda et al. 1997),whereas Arx determines α-cell identity (Collombat et al. 2003, 2005). Forced expression of Pax4,Pdx1, or Nkx6.1 in endocrine precursors favors the β-cell fate at the expense of α-cells (Collombatet al. 2009, Schaffer et al. 2013, Yang et al. 2011). Conversely, Arx gain of function results inexcess α-cells and reduced β-cells (Collombat et al. 2007). Loss-of-function experiments revealedreciprocal phenotypes, although the consequences of Pdx1 deletion in endocrine precursors areyet to be explored. Both Pax4 and Nkx6.1 are direct transcriptional repressors of Arx; conversely,Arx represses Pax4, Nkx6.1, and Pdx1 by direct or indirect mechanisms (Figure 3d ) (Collombatet al. 2005, 2007; Schaffer et al. 2013; Yang et al. 2011).

One interesting but mechanistically poorly understood observation is that some transcriptionfactors, which can change the lineage identity of endocrine precursors, lose this ability when mis-expressed in differentiated endocrine cells. For example, Pdx1 or Nkx6.1 is insufficient to convertdifferentiated α-cells into β-cells (Schaffer et al. 2013, Yang et al. 2011). This suggests that en-docrine differentiation restrains developmental plasticity. The molecular mechanisms restrictingcell plasticity during cell differentiation are poorly understood. Recent studies indicate a role forDNA methylation (Dhawan et al. 2011, Papizan et al. 2011), but additional studies are needed tounderstand how such epigenetic mechanisms limit cell plasticity. A better understanding of thesemechanisms could enable strategies for reversing epigenetic constraints in efforts to reprogramnon-β endocrine cells into β-cells.

PANCREAS MORPHOGENESIS

Coincident with the differentiation of endocrine and exocrine cells, the pancreas develops into ahighly branched organ, wherein groups of acinar cells coalesce around a small ductal lumen to formfunctional exocrine secretory units, and ductal cells organize into the tubular network of the pan-creatic ductal system (Pictet et al. 1972). This process, called branching morphogenesis (Figure 2),is driven by dynamic cell rearrangements and spatiotemporally controlled cell proliferation.

Although pancreatic cell differentiation and its molecular control have been studied sincethe 1990s, knowledge of how the pancreas acquires its final shape and becomes a functionalorgan is still rudimentary. Two landmark studies describing the cellular changes associated withpancreatic tubulogenesis (Kesavan et al. 2009, Villasenor et al. 2010) now provide the frameworkfor exploration of the mechanisms governing pancreatic branching morphogenesis. Prior to theinitiation of epithelial branching at around e12, the pancreatic buds form a multilayered core ofunpolarized cells engulfed by a single basement membrane abutting the mesenchyme (Figure 2a)(Hick et al. 2009, Kesavan et al. 2009, Villasenor et al. 2010). Pancreatic tubulogenesis begins withindividual cells acquiring apicobasal polarity: Cells develop a defined apical membrane that faces acentral lumen and a basal surface attached to a layer of extracellular matrix (ECM) (Kesavan et al.2009, Villasenor et al. 2010). Groups of newly polarized cells then form rosettes, wherein adjacentcells are linked by intercellular junctional complexes around a nascent central lumen (Figure 2b,inset) (Villasenor et al. 2010).

Newly formed microlumens are initially unconnected but eventually fuse into a luminal plexus(Figure 2c). Remodeling of the luminal plexus produces a tubular network lined by a polarized,monolayered epithelium attached to a basement membrane (Figure 2c, inset) (Kesavan et al.2009, Villasenor et al. 2010). At birth, the tubes have organized into a highly branched ductaltree, connecting exocrine secretory units to the ductal system (Figure 2d, inset). Concomitant toepithelial branching, blood vessels and nerves also begin to penetrate the epithelium, intercalat-ing between nascent branches of the pancreatic ductal tree (Figure 2b–d ) (Lindsay et al. 2006,


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Magenheim et al. 2011a, Pierreux et al. 2010). The stereotypical organization of cells in themature pancreas illustrates that proper pancreas morphogenesis requires highly coordinated cellrearrangements involving acinar, ductal, endocrine, endothelial, neuronal, and mesenchymal cells.Currently, little is known about the molecular cues governing these cell rearrangements; about howpancreas morphogenesis is coordinated with cell differentiation; or about how differentiation ofpancreatic endocrine and exocrine cells influences surrounding nonepithelial cells, and vice versa.

Intracellular Signals Governing Pancreas Morphogenesis

In many organs, including pancreas, epithelial morphogenesis is controlled by Rho familyGTPases, which regulate cell polarity, adhesion, and migration by organizing the actin cytoskele-ton (Heasman & Ridley 2008). Kesavan et al. (2009) demonstrated that deletion of the Rho GTPaseCdc42 interrupts cell polarization during early morphogenesis, disrupting epithelial morphologyand aborting tubulogenesis at microlumen formation. Premature arrest of tubulogenesis was alsoobserved in mice deficient in RhoA GTPase inhibitor Strad13 (Petzold et al. 2013), whereaspancreas-specific deletion of Rho GTPase Rac1 had no obvious effect on pancreas morphogenesis(Heid et al. 2011). This implies that different Rho GTPases have distinct roles in shaping the organ.

Studies in other organs show that Rho GTPases are regulated by Ephrin, EGF, and ECM-integrin signaling (Heasman & Ridley 2008, Huveneers & Danen 2009, Poliakov et al. 2004).The hypobranched pancreas seen in Ephrin and EGF receptor–mutant mice suggests that thesesignaling pathways could also influence Rho GTPase activity in the pancreas (Miettinen et al. 2000,Villasenor et al. 2010). How extrinsic cues coordinate epithelial morphogenesis and differentiationin a spatiotemporally controlled manner is an active area of research.

Coordinating Morphogenesis and Cell Differentiation

Evidence is emerging that mesenchymal cells, blood vessels, and ECM provide locally restrictedsignals that influence morphogenesis and cell fate decisions in the developing pancreas. Tip/trunksegregation not only marks the first cell fate choice of pancreatic progenitors but also coincideswith the formation of branches in the tip region (Figure 2b, inset) (Zhou et al. 2007). The tissueremodeling associated with branch formation results in the repositioning of mesenchymal andepithelial cells and basement membrane components (Kesavan et al. 2009, Landsman et al. 2011).Real-time imaging and static analysis of pancreas morphogenesis suggest that new branches formby tip-splitting (i.e., individual tips repeatedly split into new tips) (Puri & Hebrok 2007, Villasenoret al. 2010). Whether this is driven directly by differential proliferation of tip cells or indirectly bycell remodeling in the trunk is unknown. However, tip cells have been observed to proliferate morequickly than trunk cells (Petzold et al. 2013, Zhou et al. 2007), which implies that tip proliferationcould drive branch formation. One candidate cue for regulating tip-cell proliferation is FGF, whichis secreted by mesenchymal cells that surround the tips when branching is initiated (Bhushan et al.2001). Removal of mesenchyme as an FGF source halts morphogenesis of pancreatic explants,whereas adding FGF to cultures restores branching and cell proliferation (Miralles et al. 1999).

Similar to their association with mesenchymal cells, emerging tip cells are also directly ap-posed to the basement membrane at the beginning of epithelial branching (Kesavan et al. 2009,Villasenor et al. 2010). Explant culture studies have shown that knockdown or blocking of an-tibodies against the ECM component laminin-1 reduces expression of early acinar cell markersand impairs branching (Kesavan et al. 2009, Li et al. 2004). Because the branching process leadsto increased cellular exposure to ECM, the process of morphogenesis as such could cause cells toadopt acinar identity.


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Whereas mesenchymal cells and ECM components favor acinar identity and promote branch-ing, blood vessels have the opposite effect. Hypervascularization of pancreatic progenitors byforced VEGFA expression results in increased expression of the trunk marker Hes1 and con-comitant loss of the tip/acinar markers Cpa and amylase, whereas VEGF ablation favors acinarformation and causes hyperbranching (Magenheim et al. 2011a, Pierreux et al. 2010). Consistentwith their trunk-promoting effect, blood vessels remain physically associated with the trunk andbecome separated from the tips (Magenheim et al. 2011a, Pierreux et al. 2010) (Figure 2b–c,inset). These examples illustrate that organ morphogenesis and cell differentiation are intricatelylinked. Further studies should focus on identifying the extrinsic and intrinsic cues mediating crosstalk between endothelial, mesenchymal, and epithelial cells and on deciphering how these cuesgovern morphogenesis and cell differentiation. To analyze spatiotemporally dynamic intercellu-lar interactions more precisely, it would be helpful to develop a system for studying pancreasorganogenesis in vitro under defined conditions. A stem cell–based 3-D differentiation systemcould enable significant progress.

Morphogenetic Changes and Endocrine Cell Differentiation

Morphogenetic changes are also associated with the initiation of endocrine differentiation in theprogenitor cords. As cells initiate Ngn3 expression, they acquire a droplet shape and delaminatefrom the progenitor epithelium (Figure 2c, inset) (Gouzi et al. 2011, Rukstalis & Habener 2007).Gouzi et al. (2011) have shown that expression of Snail2, a protein that induces cell motilityby triggering epithelial-to-mesenchymal transition (EMT) (Barrallo-Gimeno & Nieto 2005), isNgn3 dependent. Ectopic Ngn3 expression also triggers EMT, which suggests a dual role forNgn3 in inducing both cell differentiation and delamination (Gouzi et al. 2011). The extent towhich these processes are interdependent is unknown. Conditional inactivation of Snail2, or ofother effectors of EMT, may explain the connection between endocrine cell differentiation anddelamination. Another mechanism that could enable cell delamination is asymmetric cell division,in which cells divide so that only one daughter cell inherits the apical membrane and remains inthe progenitor epithelium, while the daughter cell inheriting the basal membrane escapes from theepithelial sheath and differentiates. This mechanism is associated with the asymmetric distributionof cell fate determinants to the daughter cells and is relevant for the differentiation of neuronsand epidermal cells (reviewed in Knoblich 2008). It is unknown whether endocrine precursors arecreated by asymmetric cell divisions in progenitor cords. The knowledge may be important fordevising appropriate in vitro culture conditions for production of functional endocrine cells fromhPSCs. Rapidly improving real-time imaging technology, which now allows for the visualizationof fluorescently labeled cells in whole organ explants at single-cell resolution, will likely proveuseful for defining the cellular events associated with the emergence of endocrine precursors fromthe progenitor cords.

APPLICATION OF DEVELOPMENTAL PRINCIPLES TOWARD STEMCELL DIFFERENTIATION

The ultimate goal of understanding pancreas development is to gain insight into pancreas-associated diseases and to develop novel therapies to treat such diseases. With the knowledgeamassed by studying pancreas development, investigators are exploring novel therapeutic avenuesfor treating diabetes by replacing or regenerating β-cell mass. Such innovative strategies fordiabetes treatment include increasing β-cell numbers by boosting their intrinsic ability toreplicate; fate-converting other pancreatic cells into β-cells (reviewed in Desgraz et al. 2011);


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and most fascinatingly, both as a therapy and as a tool for study, generating β-cells in vitro bydifferentiating hPSCs.

Generating Pancreatic Progenitors from Human Pluripotent Stem Cells

The enthusiasm for deriving transplantable β-cells from hPSCs is the direct result of landmarkachievements in cell transplantation, in particular the successful allogenic transplantation of islets,which has allowed diabetic patients to forego insulin injections for years (Shapiro et al. 2006).Unfortunately, the amount of donor islets required for widespread treatment significantly exceedsavailability, and even when donor islets are available, recipients must undergo lifelong immuno-suppression to prevent rejection. Although these limitations have prevented its widespread use indiabetic patients, the insulin independence achieved with islet transplantation provides proof thatcell transplantation can be a viable therapy for diabetes. Unlike allografts, which are susceptibleto donor shortages, hPSCs could yield an unlimited source of transplantable β-cells. Addition-ally, patient-derived induced pluripotent stem cells would obviate immunosuppression. For thesereasons, there is currently a major push to generate pancreatic β-cells in vitro.

Protocols have been established for deriving pancreatic precursors from hPSCs. hPSCs canbe differentiated with high efficiency to definitive endoderm by adding high levels of Activin A,mimicking the proendodermal role of Nodal during in vivo gastrulation (reviewed by Zorn & Wells2009). A landmark study by D’Amour et al. (2006) established a protocol to guide hPSC-derivedendoderm toward the pancreatic lineage. Similar protocols have since been published (Kroon et al.2008, Mfopou et al. 2010, Nostro et al. 2011, Rezania et al. 2012, Xu et al. 2011). The majorityof pancreatic differentiation protocols induce pancreatic fate by simultaneously treating cells withRA and inhibitors of Shh and BMP, recapitulating the signaling environment that specifies thepancreas in vivo. Such treatment generates the in vitro equivalent of early multipotent pancreaticprogenitors, expressing PDX1, SOX9, PTF1a, and NKX6.1. These in vitro–generated progenitorsalso pass the functional test of having the potential to form mature endocrine, ductal, and acinarcells when transplanted into mice (Kelly et al. 2011, Kroon et al. 2008, Rezania et al. 2012, Schulzet al. 2012, Xie et al. 2013). The resulting endocrine cells are functional, responding appropriatelyto glucose and properly regulating glucose homeostasis in diabetic animal models (Kroon et al.2008, Rezania et al. 2012, Xie et al. 2013). Further evidence for their islet-like phenotype is thatthe transcriptional signature and cellular composition of hPSC-derived endocrine clusters arestrikingly similar to those of human islets (Xie et al. 2013).

Having accomplished the in vitro differentiation of functional pancreatic progenitors, the fieldhas attempted to optimize differentiation efficiencies across different cell lines. In addition, to allowfor large-scale cell production, strategies have been developed to expand developmental lineageintermediates in vitro (Cheng et al. 2012, Schulz et al. 2012, Sneddon et al. 2012). However,because it is unknown whether transplantation of pancreatic progenitors can cure diabetes inhumans, the ultimate goal is to generate functional β-cells in vitro.

Beyond Progenitors: How to Generate Functional Endocrine Cells In Vitro

Currently, endocrine cells that arise in vitro are non–glucose responsive and often coexpressmultiple hormones. For pancreatic differentiation of hPSCs to fulfill its full therapeutic andscientific promise, methods to generate functional pancreatic cells in vitro must be developed.As discussed in the sections on Lineage Decisions During Pancreas Development and PancreasMorphogenesis, above, the developing pancreas receives extrinsic signals from the mesenchyme,ECM, and endothelium. However, these components are not supplied in the majority of hPSC


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differentiation protocols. Coculture with mesenchymal cell lines has been explored as a strategyto expand intermediate precursor populations, consistent with the role of mesenchyme in earlypancreatic progenitor expansion (Sneddon et al. 2012). Possible instructive signals from the mes-enchyme have been added to current pancreatic differentiation protocols and have resulted inincreased efficiency of PDX1 induction (Guo et al. 2013). The role of mesenchyme and ECM atlater steps of pancreatic development in vivo, especially during endocrine cell differentiation, isstill poorly understood. Both should be considered as a possible source for signals when developingstrategies to generate functional β-cells in vitro.

Blood vessels may also be involved in islet development, because both native islets and in vivo–matured, transplanted hPSC-derived endocrine cells are highly vascularized with endocrine cellsin direct contact with blood vessels. Although endothelial cells are clearly important for propersensing of blood glucose levels and release of hormones, their role in pancreatic lineage determi-nation is only beginning to be unveiled. Because blood vessels promote trunk cell formation atthe expense of tip cell formation (see section on Coordinating Morphogenesis and Cell Differ-entiation) (Magenheim et al. 2011a, Pierreux et al. 2010), endothelial cells could provide criticalmissing cues for favoring the endocrine cell fate during in vitro differentiation. As discussed in thesection on Pancreas Morphogenesis, the embryonic pancreas undergoes extensive morphogeneticchanges in a 3-D epithelium, which may be coupled with cell-lineage decisions. Unlike in vivodevelopment, in vitro pancreatic differentiation of hPSCs is primarily performed in 2-D culturesystems. This difference between in vitro and in vivo differentiation conditions could, at least inpart, explain our inability to generate functional β-cells in vitro. Protocols to generate functionalcells of other endodermal lineages (e.g., the intestine) have employed 3-D culture systems. Such3-D systems support proper morphogenesis and have been shown to produce mini organs with re-markable similarity to their in vivo counterparts (Sato et al. 2009, Spence et al. 2011). Organotypicculture systems may also facilitate the production of functional β-cells from hPSCs.

An important question is whether the generation of islets or of only β-cells should be attempted.Engrafted hPSC-derived progenitors differentiate into functional islet-like clusters that containall endocrine cell types, in a ratio similar to that in human islets (Xie et al. 2013). Although it iscurrently unknown whether non-β islet cells are required for β-cell function, evidence suggeststhat paracrine signaling between endocrine cell types may be important in humans (reviewed inCaicedo 2012). Therefore, future studies should consider the production of entire islets and notjust of β-cells.

Human Pluripotent Stem Cells as a Model for UnderstandingPancreas Biology and Disease

Much information about pancreas development and organogenesis has been gleaned from ani-mal models; there are, however, some limitations to these models. At a technical level, transientdevelopmental cell populations are too sparse in embryos to isolate sufficient cells for large-scalegenomic or biochemical analyses. The ability to differentiate such lineage intermediates fromhPSC in virtually unlimited quantities has enabled such studies and recently has led to the iden-tification of core transcriptional and epigenetic mechanisms of pancreatic lineage determination(Xie et al. 2013).

Moreover, species-specific differences in gene regulation limit the use of animal models forstudying human disease. One example is maturity onset diabetes of the young (MODY), which inhumans results from single-gene mutations that are not always phenocopied in mice (Haumaitreet al. 2006, Mayer et al. 2008). It is hoped that protocols for the in vitro production of functionalβ-cells will facilitate studies aimed at understanding disease mechanisms of diabetes. Advances


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in genome editing now allow us to model effects of gene mutations or single-nucleotide poly-morphisms in hPSCs. Thus, the production of β-cells in vitro not only will be a significant steptoward regenerative medicine but also will help advance our understanding of the pathogenesisof diabetes.

FUTURE PROSPECTS

This review covers the tremendous progress the field has made in understanding pancreaticorganogenesis and briefly discusses the progress made in recapitulating this process in vitro usinghPSCs. Reflecting on this body of work, a few conclusions become strikingly clear. First, muchremains to be understood about the complex process of pancreas organogenesis, specifically, howmorphogenesis is coupled with cell fate specification. Although we have made significant progressin understanding many of the transcription factors that are required at various stages of pancreasdevelopment, the signals that promote and regulate each developmental decision are still poorlyunderstood. Clearly, the current success of in vitro pancreatic differentiation of hPSCs has reliedheavily on studies employing animal models. Therefore, we must continue to apply the lessonslearned in vivo to achieve the critical goal of generating functional β-cells in vitro.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank members of the Sander laboratory for constructive comments on the manuscript. Weapologize to our colleagues whose references were omitted owing to space constraints. Work inthe Sander laboratory is supported by grants from the National Institutes of Health (NIH), theJuvenile Diabetes Research Foundation, and the California Institute for Regenerative Medicine.H.P.S. and A.W. were supported by postdoctoral fellowships from the Juvenile Diabetes ResearchFoundation. A.W. was additionally supported by the NIH training program in diabetes researchT32 DK7494-27.

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CB29-FrontMatter ARI 16 September 2013 12:50

Annual Reviewof Cell andDevelopmentalBiology

Volume 29, 2013 Contents

Formation and Segmentation of the Vertebrate Body AxisBertrand Benazeraf and Olivier Pourquie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Mechanobiology and Developmental ControlTadanori Mammoto, Akiko Mammoto, and Donald E. Ingber � � � � � � � � � � � � � � � � � � � � � � � � � � � �27

Mitogen-Activated Protein Kinase Pathways in OsteoblastsMatthew B. Greenblatt, Jae-Hyuck Shim, and Laurie H. Glimcher � � � � � � � � � � � � � � � � � � � � � �63

Pancreas Organogenesis: From Lineage Determinationto MorphogenesisHung Ping Shih, Allen Wang, and Maike Sander � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �81

Beyond the Niche: Tissue-Level Coordination of Stem Cell DynamicsLucy Erin O’Brien and David Bilder � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 107

Hormonal Control of Stem Cell SystemsDana Gancz and Lilach Gilboa � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 137

Spermatogonial Stem Cell Self-Renewal and DevelopmentMito Kanatsu-Shinohara and Takashi Shinohara � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 163

Kin Conflict in Seed Development: An Interdependentbut Fractious CollectiveDavid Haig � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

microRNA Control of Mouse and Human Pluripotent StemCell BehaviorTobias S. Greve, Robert L. Judson, and Robert Blelloch � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 213

Something Silent This Way Forms: The Functional Organizationof the Repressive Nuclear CompartmentJoan C. Ritland Politz, David Scalzo, and Mark Groudine � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 241

Cytoskeletal Dynamics in Caenorhabditis elegans Axon RegenerationAndrew D. Chisholm � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 271

viii

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CB29-FrontMatter ARI 16 September 2013 12:50

Integrative Mechanisms of Oriented Neuronal Migration in theDeveloping BrainIrina Evsyukova, Charlotte Plestant, and E.S. Anton � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 299

TRP Channels and PainDavid Julius � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 355

Synaptic Laminae in the Visual System: Molecular MechanismsForming Layers of PerceptionHerwig Baier � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 385

Microtubule-Depolymerizing KinesinsClaire E. Walczak, Sophia Gayek, and Ryoma Ohi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 417

Kinesin-2: A Family of Heterotrimeric and Homodimeric Motorswith Diverse Intracellular Transport FunctionsJonathan M. Scholey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 443

Microtubules in Cell MigrationSandrine Etienne-Manneville � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 471

Mathematical Modeling of Eukaryotic Cell Migration:Insights Beyond ExperimentsGaudenz Danuser, Jun Allard, and Alex Mogilner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 501

GTP-Dependent Membrane FusionJames A. McNew, Holger Sondermann, Tina Lee, Mike Stern,

and Federica Brandizzi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 529

Viral Membrane ScissionJeremy S. Rossman and Robert A. Lamb � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 551

How Microbiomes Influence Metazoan Development: Insights fromHistory and Drosophila Modeling of Gut-Microbe InteractionsWon-Jae Lee and Paul T. Brey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 571

Cell and Developmental Biology of Arbuscular Mycorrhiza SymbiosisCaroline Gutjahr and Martin Parniske � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 593

Indexes

Cumulative Index of Contributing Authors, Volumes 25–29 � � � � � � � � � � � � � � � � � � � � � � � � � � � 619

Cumulative Index of Article Titles, Volumes 25–29 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 622

Errata

An online log of corrections to Annual Review of Cell and Developmental Biology articlesmay be found at http://cellbio.annualreviews.org/errata.shtml

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ANNUAL REVIEWSIt’s about time. Your time. It’s time well spent.

ANNUAL REVIEWS | Connect With Our ExpertsTel: 800.523.8635 (US/CAN) | Tel: 650.493.4400 | Fax: 650.424.0910 | Email: [email protected]

New From Annual Reviews:

Annual Review of Statistics and Its ApplicationVolume 1 • Online January 2014 • http://statistics.annualreviews.org

Editor: Stephen E. Fienberg, Carnegie Mellon UniversityAssociate Editors: Nancy Reid, University of Toronto

Stephen M. Stigler, University of ChicagoThe Annual Review of Statistics and Its Application aims to inform statisticians and quantitative methodologists, as well as all scientists and users of statistics about major methodological advances and the computational tools that allow for their implementation. It will include developments in the fi eld of statistics, including theoretical statistical underpinnings of new methodology, as well as developments in specifi c application domains such as biostatistics and bioinformatics, economics, machine learning, psychology, sociology, and aspects of the physical sciences.

Complimentary online access to the fi rst volume will be available until January 2015. TABLE OF CONTENTS:• What Is Statistics? Stephen E. Fienberg• A Systematic Statistical Approach to Evaluating Evidence

from Observational Studies, David Madigan, Paul E. Stang, Jesse A. Berlin, Martijn Schuemie, J. Marc Overhage, Marc A. Suchard, Bill Dumouchel, Abraham G. Hartzema, Patrick B. Ryan

• The Role of Statistics in the Discovery of a Higgs Boson, David A. van Dyk

• Brain Imaging Analysis, F. DuBois Bowman• Statistics and Climate, Peter Guttorp• Climate Simulators and Climate Projections,

Jonathan Rougier, Michael Goldstein• Probabilistic Forecasting, Tilmann Gneiting,

Matthias Katzfuss• Bayesian Computational Tools, Christian P. Robert• Bayesian Computation Via Markov Chain Monte Carlo,

Radu V. Craiu, Jeff rey S. Rosenthal• Build, Compute, Critique, Repeat: Data Analysis with Latent

Variable Models, David M. Blei• Structured Regularizers for High-Dimensional Problems:

Statistical and Computational Issues, Martin J. Wainwright

• High-Dimensional Statistics with a View Toward Applications in Biology, Peter Bühlmann, Markus Kalisch, Lukas Meier

• Next-Generation Statistical Genetics: Modeling, Penalization, and Optimization in High-Dimensional Data, Kenneth Lange, Jeanette C. Papp, Janet S. Sinsheimer, Eric M. Sobel

• Breaking Bad: Two Decades of Life-Course Data Analysis in Criminology, Developmental Psychology, and Beyond, Elena A. Erosheva, Ross L. Matsueda, Donatello Telesca

• Event History Analysis, Niels Keiding• Statistical Evaluation of Forensic DNA Profi le Evidence,

Christopher D. Steele, David J. Balding• Using League Table Rankings in Public Policy Formation:

Statistical Issues, Harvey Goldstein• Statistical Ecology, Ruth King• Estimating the Number of Species in Microbial Diversity

Studies, John Bunge, Amy Willis, Fiona Walsh• Dynamic Treatment Regimes, Bibhas Chakraborty,

Susan A. Murphy• Statistics and Related Topics in Single-Molecule Biophysics,

Hong Qian, S.C. Kou• Statistics and Quantitative Risk Management for Banking

and Insurance, Paul Embrechts, Marius Hofert

Access this and all other Annual Reviews journals via your institution at www.annualreviews.org.

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Documents

Pancreas Organogenesis: From Lineage …msanderlab.org/wp-content/uploads/2017/07/Shih-2013...the cardiac mesoderm surrounding the prehepatic region (Deutsch et al. 2001, Rossi et