Analysis of the differentiation potential of murine pancreatic islet precursor cells: an In Vivo clonal analysis

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Thesis

Reference

Analysis of the differentiation potential of murine pancreatic islet precursor cells: an In Vivo clonal analysis

DESGRAZ, Renaud

Abstract

Le pancréas est une glande mixte du système digestif. La partie exocrine (~99%) est responsable de la production et sécrétion d'enzymes digestives. La partie endocrine (~1%) se compose de quatre types cellulaires : α, β, δ et PP, assemblés en "îlots de Langerhans", qui synthétisent et sécrètent des hormones régulant l'appétit, la motilité gastrique et la glycémie.

Des études de traçage cellulaire ont montré que ces quatre types cellulaires endocrines dérivent de cellules exprimant le gène Neurogenin3. Afin de déterminer le potentiel de différentiation (unipotent ou multipotent) des cellules exprimant la Neurogenin3, nous avons procédé à une analyse clonale in vivo de celles-ci. Nous avons établis que ces cellules à Neurogenin3 représentent une population hétérogène de précurseurs unipotents. Plus particulièrement, ces résultats impliquent que les cellules β, productrices d'insuline, se différencient à partir de cellules Neurogenin3+ spécifiques, et ne prolifèrent que très peu au cours de la vie.

DESGRAZ, Renaud. Analysis of the differentiation potential of murine pancreatic islet precursor cells: an In Vivo clonal analysis. Thèse de doctorat : Univ. Genève, 2009, no. Sc.

4125

URN : urn:nbn:ch:unige-45652

DOI : 10.13097/archive-ouverte/unige:4565

Available at:

http://archive-ouverte.unige.ch/unige:4565

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITE DE GENEVE

Département de FACULTÉ DES SCIENCES

zoologie et biologie animale Professeur Iván Rodríguez

Département de FACULTÉ DE MÉDECINE

médecine génétique et développement Professeur Pedro L Herrera ___________________________________________________________________

Analysis of the Differentiation Potential of Murine

Pancreatic Islet Precursor Cells: an In Vivo Clonal Analysis

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

Par

Renaud DESGRAZ de

Puidoux (VD)

Thèse N° 4125

GENÈVE ReproMail

2009

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Acknowledgments

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First of all I would like to thank Professor Pedro L Herrera for giving me the opportunity to do my PhD in his laboratory. I would like to thank him for wisely guiding me in the tortuous path of science and for creating an enthusiastic and humane scientific environment.

I would like to thank Professor Christopher Wright and Professor Iván Rodríguez, for accepting to be members of my thesis jury.

I am deeply grateful to Gissela Flores without whom this work would not have been possible, for her skilful help with the experiments and for her funky attitude in the lab.

I want to thank very specially Claire Bonal who struggled alongside me during all these years. Her scientific advice and support was invaluable, and her enthusiasm for science and life in general was a constant source of motivation.

I thank all past and present members of the laboratory with whom I had the chance to interact: Fabrizio Thorel, Isabelle Avril, Berivan Polat, Polat Percin Olivier Fazio, Carine Gysler, Virginie Népote, Alessandra Strom, Alexandra Solomos, Dario Sessa Simona Chera and Alexandra Delacour. I thank them for their help and support and for contributing to a great working environment.

I would like also to than Prof Jean-Dominique Vassalli and Serge Nef for sharing their enthusiasm for science and for their constant support.

I would like to thank the members of the Vassalli and Nefs laboratory for scientific discussion and frenzied coffee breaks.

Finally I would like to thank my family and friends for their support and for being there when I needed them most.

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I Résumé en français

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Le pancréas est une glande annexée au tube digestif. On distingue deux parties, le pancréas ventral, près du duodénum et le pancréas dorsal, près de la rate.

La majeure partie de l’organe (99%) est une glande exocrine, composée de grappes acinaires situées au bout de canaux permettant de déverser les sécrétions exocrines dans le duodénum. Les sécrétions acinaires sont un mélange d’enzymes digestives (protéases, lipases, carboxypeptidases, …) libérées en réponse à l’ingestion de nourriture.

La partie endocrine (1% de l’organe) est composée de petits groupes compacts de cellules appelés îlots de Langerhans, répartis au sein du parenchyme de la glande.

Les îlots contiennent 4 types cellulaires : 75-80% des cellules de l’îlot sont des cellules β qui produisent et sécrètent de l’insuline en réponse à une élévation de la glycémie (augmentation du sucre dans le sang). L’insuline permet l’entrée et le stockage du glucose dans les tissus périphériques. Les cellules α représentant 15- 20% des cellules de l’îlot produisent et secrètent le glucagon en réponse à une hypoglycémie. Le glucagon permet de mobiliser le glucose des hépatocytes qui le stockent sous forme de glycogène. Les cellules δ (5%) secrètent la somatostatine qui a un effet inhibiteur sur les sécrétions pancréatiques et la vidange gastrique. Enfin les cellules PP (~1%) sécrètent le polypeptide pancréatique qui a un effet anorexigène (diminution de l’appétit) en réponse à l’ingestion de nourriture.

Lors du développement murin, le pancréas se forme à partir de deux bourgeonnements (l’un ventral et l’autre dorsal) de l’intestin primitif antérieur autour du 9^ème jour de développement (E9, celui-ci s’étend sur une vingtaine de jours). Les premières cellules des bourgeons pancréatiques expriment les facteurs de transcription Pdx1 et Ptf1a et sont multipotentes, c'est-à-dire qu’elles sont progénitrices de toutes les cellules épithéliales qui vont composer l’organe adulte.

Les cellules des deux bourgeons prolifèrent et se différencient puis les deux bourgeons fusionnent vers E12.5 pour former la glande mature et fonctionnelle à la naissance.

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L’apparition des cellules endocrines se fait en deux vagues, tout d’abord entre E9.5 et E12.5 (période appelée « première transition ») puis entre E13.5 et la naissance (« deuxième transition »). Alors que les cellules issues de la première transition apparaissent immatures (coexpression de plusieurs hormones dans la même cellule, absence de marqueurs de différentiation), les cellules endocrines issues de la deuxième transition sont matures. Toutes les cellules endocrines du pancréas dérivent de cellules indifférenciées exprimant le facteur de transcription Neurogenin3 (Ngn3). Ngn3 est exprimé de manière transitoire au cours du développement et sa présence dans le pancréas adulte est sujette à controverse.

Le but de cette étude est de déterminer si une cellule exprimant Ngn3 est capable de se différencier en plusieurs types endocrine pancréatiques (progéniteur multipotent) ou uniquement capable de donner un seul type (précurseur unipotent). Pour cela, nous avons réalisé une analyse clonale in vivo, reposant sur le marquage irréversible d’une infime fraction des cellules exprimant Ngn3+. Nous avons ensuite regardé la progéniture des cellules marquées dans les îlots adultes pour déterminer si le groupe de cellules composant cette descendance clonale exprimait la même hormone ou des hormones différentes. En effet, l’expression de différents types hormonaux au sein de la progéniture indiquerait que la cellule marquée était multipotente alors que la présence d’un seul type hormonal dans la progéniture indique que la cellule était unipotente.

Nous avons observé que dans les îlots en formation à la naissance, la plupart des cellules marquées étaient des cellules endocrines différenciées et isolées. Ce résultat indique que, entre le moment où ces cellules expriment Ngn3 et la naissance il n’y a pas eu de divisions dans la lignée endocrine : une cellule exprimant Ngn3+

pendant l’embryogénèse devient une cellule endocrine à la naissance. Ce résultat en soi suggère déjà fortement que les cellules exprimant Ngn3 sont unipotentes.

Nous avons ensuite regardé la composition des groupes de cellules marquées dans les îlots adultes à 2 mois et nous avons observé que la majorité (~75%) de ces groupes de cellules exprimaient une seule hormone. Ce résultat est en accord avec un modèle où les cellules Ngn3+ sont unipotentes. Toutefois, il y avait 25% des groupes de cellules marquées qui exprimait plusieurs hormones, nous avons donc exploré si ces groupes hétérogènes résultent en fait de la fusion de deux groupes

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homogènes exprimant des hormones différentes. Nous avons pu confirmer cette hypothèse mathématiquement, renforçant nos conclusions selon lesquelles les cellules Ngn3+ forment une population hétérogène de précurseurs unipotents.

Nous avons également pu montrer que l’abondance des cellules β dans l’îlot pas rapport aux autre types endocrines, n’est pas uniquement due à une prolifération supérieure mais surtout à un plus grand nombre de précurseurs engagés dans la voie de différenciation β pendant l’embryogénèse.

Il est maintenant important de déterminer quels sont les facteurs extracellulaires (voie de signalisation, facteurs de croissance) et intracellulaires (événement génétiques et épigénétiques) gouvernant la différenciation de progéniteurs pancréatiques en cellules β. Une meilleure compréhension de ces mécanismes permettra de développer de nouveaux protocoles de différentiation de cellules β in vitro afin de traiter les patients diabétiques.

Utilisant la même approche nous avons également exploré l’homéostasie des îlots entre 2 et 10 mois chez la souris. Nous avons observé qu’entre 2 et 10 mois la taille des îlots augmente : le volume moyen des îlots est 1.5 fois plus grand alors que le nombre absolu d’îlots reste constant. Dès lors, trois mécanismes peuvent contribuer à l’augmentation de la taille des îlots : la réplication de cellules insulaires préexistantes, la néogenèse à partir de cellules souches adultes et la transdifferentiation à partir de cellules pancréatiques non insulaires. En se basant sur les variations des proportions de cellules marquées au cours du temps nous avons déterminé que le mécanisme prévalent est la réplication des cellules insulaires préexistantes.

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II English Summary

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The pancreas is an amphicrine gland of the abdominal cavity. The exocrine part, representing ~99% of the organ, is formed by a tree of ducts terminated by clusters of acinar cells synthesizing and secreting digestive enzymes.

The endocrine part, called islets of Langerhans, representing ~1% of the organ, is composed by group of cells scattered throughout the organ’s parenchyma. Islets contain four different cell types: β-cells secreting insulin, α-cells glucagon, δ-cells somatostatin and PP-cells pancreatic polypeptide. A decrease in the functional β-cell mass leads to glucose intolerance and eventually diabetes, a disease affecting millions of people worldwide.

Genetic lineage tracing studies showed that all pancreatic cells derive from multipotent progenitors expressing the transcription factors Pdx1 and Ptf1a. On the other hand, islet endocrine cells arise during development from precursors expressing Neurogenin3 (Ngn3). As a population, Ngn3+ cells produce all islet cell types, but the potential of individual Ngn3+ cells, an issue central to organogenesis in general and in vitro differentiation towards cell-based therapies has not been addressed.

We performed a clonal analysis using a new genetic system to label and follow the progeny of a very small number of Ngn3-expressing islet progenitor cells. This genetic system, mosaic analysis with double markers (MADM), relies on cre- mediated inter-chromosomal recombination to reassemble a fluorescent gene and hence, label the cre-expressing cell. We then scored large numbers of progeny arising from single Ngn3+ cells to analyze the differentiation potential and proliferative capacities of endocrine cells.

In newborns, labeled islets frequently contained just one single tagged endocrine cell, indicating for the first time that each Ngn3+ cell is precursor of one single endocrine cell. In adults, clusters of labeled cells representing the progeny of individual Ngn3-expressing cells were mainly homogeneous in terms of hormone

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expression, indicating that a single Ngn3-expressing cell is unipotent. The analysis of labeled clusters at 2 and 10 months confirmed previous observations, showing that islet cell turnover is slow in adult islets and that islet cells arise mainly by proliferation of pre-existing islet cells.

These results open new questions and allow addressing fundamental aspects regarding the behavior of single, individual cells, rather than entire populations. We now need to identify the extrinsic and intrinsic factors governing β-cell specification from multipotent Pdx1-expressing cells so as to improve protocols aiming at differentiating β-cells from ES or iPS cells and promoting endogenous β-cell regeneration.

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III Introduction

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The Pancreas

The pancreas is an accessory gland of the digestive tract. It is located in the abdominal cavity retroperitoneally in humans but intraperitoneally in mice.

Anatomically, it is composed of two parts: the head of the pancreas which is attached to the duodenum and the antral stomach and the tail which extends towards the spleen. The adult murine pancreas is composed of several lobes delimited by connective tissue.

The pancreas is amphicrine, i.e. composed of exocrine and endocrine parts. The exocrine pancreas represents 95-99% of the organ. It is formed by a tree of ducts terminated by clusters of acinar cells, which produce and store zymogen granules containing a cocktail of digestive enzymes such as lipases, carboxypeptidases and proteases. Food ingestion triggers a neural (acetylcholine) and humoral (cholecystokinin, secretin or gastrin) response. These factors induce the secretion of zymogen granules which are released into lumen of acini and flushed into the duodenum via the network of tubular ducts.

The endocrine part (1-5%) is formed by the islets of Langerhans, clusters of cells scattered throughout the pancreatic parenchyma. Four different cell types compose the adult islets: α-, β-, δ- and PP-cells, secreting glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. A fifth cell type (ε-cell) appears transiently in the developing pancreas. While some ε-cells express only ghrelin, most of them also contain glucagon. Insulin producing β-cells are the most abundant (~85%) followed by α-cells (15-20%), δ-cells (5%) and PP cells (1%) (Herrera et al., 1991; Orci, 1982).

The architecture of pancreatic islets is precisely defined. In rodents, for instance, islets are formed by a core of β-cells surrounded by cells expressing other hormones.

By contrast, in human islets endocrine cells are more intermingled. Finally, the zebrafish pancreas initially harbors a single large islet to be joined somewhat later by a number of smaller islets. Pancreatic hormones are secreted into the bloodstream

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and regulate a wide range of metabolic processes such as glycemia and gastric emptying.

Pancreatic α-cells produce and secrete glucagon in response to low blood glucose levels. Glucagon stimulates glycogenolysis in hepatocytes to raise blood glucose levels. Insulin secreted by β-cells has the opposite effect: it triggers glucose entry into peripheral cells such as hepatocytes, myocytes and adipocytes. Insulin-mediated glucose entry into hepatocytes and adipocytes triggers glycogen and fatty acid synthesis, respectively. Somatostatin released by δ-cells inhibits the pancreatic exocrine and endocrine secretions, as well as the release of gastrointestinal peptides and gastric emptying. Finally, pancreatic polypeptide is secreted in PP-cells in response to food ingestion and has an anorexigenic effect reducing food intake in mice and humans (reviewed in (Hameed et al., 2009)).

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Early murine development and endodermal germ layer formation

Fertilization and implantation of the mammalian embryo

In mammals, fertilization of the ovum by the spermatozoa takes place in the ampulla of the oviduct. Soon after fertilization, the zygote surrounded by the zona pellucid and corona radiata starts to cleave, forming a multi-celled structure called the blastocyst. Upon reaching the uterus, the blastocyst hatches from the zona pellucida and adheres to the uterine wall through integrins present at the membrane of the trophoblast cells that will attach to the uterine collagen. The implantation then relies on trophoblastic proteases that digest the extracellular matrix of the uterine tissue, burying the embryo within the uterine wall. Implantation is necessary for the generation of extraembryonic tissues which will support and protect the embryo. At this stage (embryonic day 4.5 (E4.5)), the late blastocyst stage embryo is characterized by three restricted lineage cell populations (Figure 1). The trophectoderm, mediating implantation and placentation, the extraembryonic (visceral) endoderm, which essentially develops extra-embryonic structures supporting the development of the embryo proper, and the epiblast, which will give rise to both the somatic and the germ lineages of the embryo. This model has recently been challenged in a study showing that extraembryonic endodermal cells (primitive endoderm) also contribute to the formation of the definitive endoderm (Kwon et al., 2008).

Figure 1 Schematic of a blastocyst, modified from (Tam and Loebel, 2007)

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Gastrulation starts around E6.5 by the formation of the primitive streak and Hensen’s node. A proportion of epiblast cells migrate through the primitive streak to form the mesoderm and endoderm. Epiblast cell that do not pass through the primitive streak form the ectoderm. Ectoderm, mesoderm and endoderm constitute the three primitive germ layers (Figure 2). Once established, cells from a given germ layer cannot contribute to the formation of organs derived from another germ layer and so, at this stage, their fate is already becoming restricted. The endoderm forms the linings of two tubular systems within the embryo, the digestive tube and respiratory tubes (Gilbert, 2000; Tam and Loebel, 2007).

Figure 2 Murine embryo at E6.5 depicting the early primitive streak at the onset of gastrulation, modified from (Tam and Loebel, 2007)

Endoderm patterning occurs during gastrulation. Epiblast cells entering the node first (in the distal primitive streak) will form the anterior endoderm and so on. Lineage tracing analyses showed that cells expressing Hex (Hematopoietically expressed homeobox) enter the node first and colonize the ventral foregut whereas cells expressing FoxA2 form the dorsal foregut, midgut and eventually the hindgut (Lawson et al., 1991). Proper patterning of the endoderm is characterized by the specific expression of transcription factors along the Antero-Posterior axis (Grapin- Botton, 2005). At the cellular level, endoderm patterning is orchestrated by soluble factors and cell-cell contacts. For instance low β-catenin in the anterior endoderm

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maintains a foregut state whereas high β-catenin levels in the posterior promote intestinal development (McLin et al., 2007).

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Specification of endodermal cells into pancreatic progenitors.

Sonic hedgehog (Shh) signaling

The mouse pancreas develops as two evaginations, ventral and dorsal, budding out of the endoderm at the foregut/midgut junction. Its developmental program is initiated by factors released by the mesoderm at E9.0 (6-10 somites). At E13.5 (56-60s) the gut rotates clockwise, bringing the two buds together; eventually, they will fuse to form the mature organ. The ventral bud develops close to the cardiac mesoderm while the dorsal bud is under the notochord. Thus, different signals initiate ventral and dorsal pancreas budding and organogenesis (Figure 3).

Figure 3 Scheme of the different cell domains and signals implicated in embryonic pancreas and liver specification (Zaret and Grompe, 2008)

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Sonic hedgehog (Shh) is expressed in the foregut, midgut and hindgut endoderm with the exception of the pancreatic endoderm. Shh acts on a membrane-bound receptor complex formed by patched (Ptch1) and Smoothened (Smo). Shh binding inhibits Ptch1-mediated repression of Smo. Smo then signals intracellularly to activate transcription factors of the Gli family which in turn will activate or repress target genes (Figure 4).

Figure 4 The basic components of the Sonic Hedgehog pathway (Owens and Watt, 2003)

The inhibition of Shh is required for dorsal bud growth and is mediated by Activin-βB, a member of the transforming growth factor-β (TGF-β) family of proteins, secreted by the notochord. Thus, dorsal bud growth requires the presence of a functional notochord. Activin signaling is mediated by two activin receptors, ActRIIA and ActRIIB. In mice mutant for these receptors Shh is expressed inappropriately in the anterior stomach and the pancreas is drastically reduced (Kim et al., 2000). FGF2 and BMP6, two other members of the TGF-β family of proteins are important for proper pancreas development, when misexpressed under the control of a Pdx1

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promoter it leads to pancreas agenesis (Dichmann et al., 2003). Similarly, in chicken embryo, suppression of endodermal expression of Shh is required to permit the expression of Pdx1. This inhibition is mediated by FGF2, a member of the fibroblast growth factor family expressed and released by the notochord (Hebrok et al., 1998;

Kim et al., 1997).

Similarly, ex vivo development and growth of the stomach and intestinal primordia in the presence of cyclopamine, an inhibitor of the Shh pathway, results in an expansion of the pancreatic domain up to the stomach and duodenum (Kim and Melton, 1998). Similarly, ectopic expression of Shh in pancreatic cells with a Pdx1 promoter leads to spleen agenesis and the conversion of the pancreas mesenchyme into smooth muscle and interstitial cells of Cajal characteristic of the intestine (Apelqvist et al., 1997).

The ventral bud arises from the ventral foregut endoderm. A gradient of FGF secreted by the cardiac mesoderm directs this bipotential region into a close high FGF area inducing Shh (liver primordia) and a distant low FGF remaining Shh negative (ventral pancreas primordia). Thus, FGF signaling from the cardiac mesoderm does not induce the liver fate but rather represses a default pancreatic program (Deutsch et al., 2001).

Onecut1 (Hnf6)

Onecut1 is a one cut domain family transcription factor. Its expression appears around E9 in the nervous system and derivatives of the endoderm. At E12.5 the pancreatic primordia expresses widely Onecut1. From E18, Onecut1 is downregulated in the forming islets, but still expressed in the exocrine pancreas (Landry et al., 1997). Onecut1^-/- mice have a hypoplastic pancreas due to retarded pancreatic specification of endodermal cells suggesting that Onecut1 acts upstream of Pdx1 in the pancreatic differentiation cascade. This has indeed been confirmed in vitro, showing that Onecut1 can bind and transactivate the Pdx1 promoter (Jacquemin et al., 2003). Similarly, Onecut1 was shown to activate the pro-endocrine gene Neurogenin3 (neurog3) (Jacquemin et al., 2000). Thus, mice overexpressing Onecut1 in the pancreatic primordia and β-cells under the Pdx1 promoter have

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hyperplastic islets: α-, δ- and PP-cells are increased in number and intermingled within the β-cell core of the islet. These observations show that Onecut1 downregulation in endocrine islet is necessary for appropriate islet morphogenesis (Gannon et al., 2000b). Finally, other members of the onecut transcription factor family appear to also play a role in pancreas development and differentiation as Onecut1 and Onecut2 double knockout mice displayed a more severe phenotype than single Onecut1 knockout mice (Vanhorenbeeck et al., 2007).

Mnx1 (HB9)

Motor neuron and pancreas homeobox 1 (Mnx1) is expressed throughout the A-P axis of the endoderm and shows a dorso-ventral asymmetry at E9.5 (high Mnx1 expression in the dorsalmost cells and low Mnx1 expression in the ventralmost cells) (Sherwood et al., 2009). At E10.5, Mnx1 expression precedes that of Pdx1 in the dorsal and is concomitant in the ventral pancreas. Its expression then decreases at E12.5 and from E17.5 on is restricted to β-cells. Surprisingly, Mnx1^-/- mice fail to develop a dorsal pancreas, whereas the ventral develops normally suggesting that Mnx1 is required for the evaginations and subsequent development of the dorsal pancreatic bud only. This defect is not due to a defective signaling of the surrounding mesenchyme as it expresses Isl1 and appear normal. Finally, newborn Mnx1^-/- ventral pancreas contain all four islet cell types but β-cells lack Glut2 and their number is reduced by 20-60% (Harrison et al., 1999; Li et al., 1999).

Hhex

At the 6-7s stage, Hematopoietically expressed homeobox (hhex) is expressed in the foregut endoderm. During this period, the ventral foregut endoderm cells constitute a leading edge of epithelium that grows ventrally. Proliferation and migration of these endodermal cells cause the curved foregut sheet to form a tube.

Proliferation of the foregut endoderm is also critical to position the prospective ventral pancreatic domain during specification and is controlled by Hhex. It allows this segment of the endoderm to avoid cardiac mesoderm-mediated hepatogenic signaling and initiate pancreatic development. In Hex^-/- mutants the definitive

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endoderm fails to grow past the cardiac mesoderm. As a result the hepatic but not the ventral pancreatic endoderm is induced, resulting in ventral pancreas agenesis (Bort et al., 2004).

Gata4 and Gata6

The early expression pattern of Gata binding proteins 4 and 6 (Gata4 and Gata6) is controversial, some claim that they are expressed throughout the developing pancreatic epithelium at E9.5 (Decker et al., 2006) while others claim that its expression is juxtaposed to the developing buds (Ketola et al., 2004). By E16.5 Gata4 expression is restricted to the acinar cells and excluded from both ducts and developing islets (Decker et al., 2006). Gata4 mutant mice lack Pdx1-expressing cells in the ventral bud region and thus fail to form a ventral pancreas (Watt et al., 2007).

Similar to Gata4, Gata6 is expressed throughout the pancreatic epithelium at E9.5.

By E12.5, Gata6 expression is restricted to ductal and endocrine progenitor cells expressing Nkx2.2 and Neurog3. Between E14.5 and E15.5, Gata6 is strongly expressed in ductal cells and disappears from the differentiating acinar cells (Decker et al., 2006). Gata6 mutant mice also lack a ventral bud but some Pdx1 expressing cells remain in the ventral bud region (Watt et al., 2007). Thus, the development of the pancreatic epithelium is compromised by the lack of Gata6 but not as severely as in the Gata4 mutants.

Pdx1

Pancreatic and duodenal homeobox 1(Pdx1) expression starts at E8.5 in the dorsal endoderm at the foregut/midgut junction. At E9, when the gut becomes tubular, Pdx1 is seen in the antral stomach, along the dorsal, lateral and ventral region of the presumptive duodenum and in cells forming the dorsal pancreas.

Shortly after at E9.5, it is induced in the ventral pancreatic primordia (Guz et al., 1995). Pdx1 is expressed early in all pancreatic progenitor cells and later restricted to adult β-cells plus a fraction of δ-cells. Lineage tracing studies have shown that Pdx1-

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expressing cells at E8.5 are multipotent progenitors, giving rise to all pancreatic lineages (Figure 5) (Bonal et al., 2009; Herrera et al., 2002).

Figure 5 Lineage tracing from E10.5 to adults with Pdx1-cre;R26R transgenic mice showing the multipotency of Pdx1-expressing progenitors (image courtesy of Claire Bonal)

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Pdx1 is absolutely necessary for the formation of the pancreas as Pdx1^-/- mice display a pancreatic agenesis and (Offield et al., 1996). Interestingly, some Insulin and Glucagon-expressing cells develop in the dorsal bud region. Mice heterozygous for Pdx1 have elevated blood glucose levels and are glucose intolerant (Ahlgren et al., 1998). Similarly, humans bearing a heterozygous mutations of Ipf1/Pdx1 are highly predisposed to mature onset diabetes (MODY) and adult-onset Type II diabetes while its complete absence results in pancreatic aplasia (Stoffers et al., 1997a; Stoffers et al., 1998; Stoffers et al., 1997b). In addition, Pdx1 knockout mice have distorted gastro-duodenal junction, loss of Brunner’s glands and deficiency of enteroendocrine differentiation in the stomach and duodenum. However, the common bile duct and intestine form normally (Jonsson et al., 1994; Offield et al., 1996). Interestingly, although the pancreatic epithelium of Pdx1^-/- mice fails to develop, the surrounding mesenchyme grows and develops normally indicating that development of the pancreatic mesenchyme does not rely on the epithelium (Ahlgren et al., 1996).

The transcriptional events governing Pdx1-dependant early pancreas development are not fully elucidated. Four different conserved sequence elements have been defined on the Pdx1 promoter (Area I, II, III, IV) (Gerrish et al., 2001; Gerrish et al., 2004). Deletion of the areas I-II-III impairs ventral bud specification but not the dorsal one, indicating that both buds have different requirements regarding Pdx1 expression (Fujitani et al., 2006). Forkhead transcription factors A1 and A2 (Foxa1, Foxa2) have been shown to bind to the area IV of the Pdx1 promoter. Double conditional knockout of Foxa1 and Foxa2 in Pdx1-expressing cells prevents Pdx1 expression, causing near total pancreatic agenesis (Gao et al., 2008). It thus seems that spatio-temporal regulation of Pdx1 expression meditated by area I to IV is critical for proper pancreas development and differentiation. Finally, it has been shown that the number of Pdx1+

pancreatic progenitors is critical for the final size of the organ. When Pdx1-expressing cells are constitutively ablated up to E12.5, the developing pancreas does not show any growth compensation and the relative weight of the pancreas is significantly decreased at birth. These results suggest that the pool of progenitors is somehow fixed at E12.5 and increased proliferation cannot compensate for an early loss of these progenitors. Conversely, when similar experiments are performed during liver development, there is no difference in liver weight at birth (Stanger et al., 2007).

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- 22 - Ptf1 complex

Pancreas specific transcription factor-1 (Ptf1) is a trimeric complex formed by three members of the basic helix-loop-helix (bHLH) family of proteins. The first member, p75, is required to import the trimeric complex into the nucleus (Sommer et al., 1991). The two other members, p64 and Ptf1a-p48 are the DNA-binding subunits (Roux et al., 1989).

Ptf1-p48 (Ptf1a) is expressed and restricted to all pancreatic progenitors being more specific than Pdx1 which in addition to the developing pancreas is also expressed in the caudal stomach and duodenum. The coexpression of both Pdx1 and Ptf1a is necessary and sufficient to trigger the pancreatic differentiation cascade. In Xenopus, when Ptf1a is expressed ectopically in the Pdx1-expression domain it results in the formation of a giant pancreas (Afelik et al., 2006). During embryogenesis, Ptf1a expression disappears from forming islet and ductal cells at E12.5 to be restricted and superinduced in forming acinar cells during the secondary transition (Masui et al., 2007). Functional binding sites for the Ptf1complex have been found in the promoters of all acinar digestive enzyme examined (Cockell et al., 1989; Rose and MacDonald, 1997) and its role in acinar development will be discussed below. In Ptf1a-deficient embryos, pancreas development is severely impaired. Acini and ducts do not form because exocrine progenitors are reprogrammed into a duodenal fate. In these mutant mice, endocrine cells were found to be relocated into the spleen.

Surprisingly, lineage tracing analysis showed that these endocrine cells do not derive from precursors having expressed Ptf1a (Kawaguchi et al., 2002). A recent report also contest the existence of splenic endocrine cells but found β-cells expressing Mafa in the dorsal pancreatic remnant of Ptf1 mutant mice (Burlison et al., 2008).

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- 23 - Pbx1 and Isl1

Pre B-cell leukemia transcription factor 1 (Pbx1) is expressed at E10.5 in mesenchymal and Pdx1-expressing epithelial cells of the developing pancreas. In the adult it is widely expressed in ductal, acinar and islet cells. Pbx1^-/- mice exhibit agenesis of both dorsal and ventral pancreas due to a strong (35-40%) reduction of epithelial cell proliferation suggesting that Pbx1 is essential for pancreatic epithelial cell proliferation. Islet1 (isl1) and neurogenin3 (neurog3) two critical genes for the development of the endocrine cells of the pancreas are absent in Pbx1 mutant mice and accordingly no endocrine cells are present (Kim et al., 2002). In addition to controlling the proliferation of pancreatic progenitor cells, Pbx1 seems to also play a role in the differentiation of endocrine cells.

Wnt/β-catenin signaling

This family of secreted glycoproteins binds to transmembrane receptors of the Frizzled class. The best characterized pathway is the so-called “canonical” Wnt pathway. The canonical Wnt/β-catenin signaling pathway can be either “resting” or

“activated”. In the resting state, β-catenin is bound to a “destruction complex”

containing three proteins: APC, Axin and GSK3. Phosphorylation of β-catenin by GSK3 triggers its ubiquitination and degradation by the proteasome. Binding of Wnt to its receptor (Frizzled) and co-receptor (LRP5/6) activates the cytoplasmic Disheveled protein (Dsh), which disrupts the destruction complex leading to an accumulation of phophorylated β-catenin. When phosphorylated, β-catenin translocates to the nucleus and associates with DNA-binding proteins of the TCF family, activating the expression of downstream target genes such as cMyc (Figure 6).

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Figure 6 the canonical Wnt/β-catenin pathway (Moon et al., 2004)

Multiple members of the Wnt and Frz family of protein are expressed during pancreas development. Ectopic expression of Wnt1 in the Pdx1-expressing posterior foregut leads to pancreas agenesis associated with an enlarged duodenum (Heller et al., 2002) suggesting that enhanced Wnt signaling perturbs early foregut development.

Similarly, the expression of a dominant negative form of the Frz receptor under the Pdx1 promoter led to pancreas hypoplasia due to decreased proliferation of the epithelium (Papadopoulou and Edlund, 2005). The final chromatin-level effector of Wnt signaling is β-catenin, thus, several groups studied the conditional inactivation of β-catenin in the pancreatic epithelium. Inactivation of β-catenin impairs early acinar proliferation and results in a smaller pancreas at birth (Heiser et al., 2006; Murtaugh et al., 2005; Wells et al., 2007). Interestingly, in these studies the endocrine pancreas develops normally. Another study reports an early pancreatitis following the inactivation of β-catenin in the developing and newborn pancreas. Surprisingly, the pancreas recovers from the pancreatitis and appears normal at 6 weeks. Unlike the other studies, however, they observed a 50% reduction of Glucagon- and Insulin- expressing cells at E14.5 and 40% reduction of big and small islets at birth

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(Dessimoz et al., 2005). These discrepancies are surprising but could perhaps be related to the use of different strains of Pdx-cre mice which do not have exactly the same penetrance and expression pattern. Together these results suggest that an active Wnt pathway is required early during pancreas development for proper acinar cell specification.

Interestingly, artificially activating Wnt pathway later during the secondary transition by artificially stabilization of β-catenin (preventing its phosphorylation and subsequent degradation) in Pdx1-expressing cells leads to pancreas hyperplasia (Heiser et al., 2006). This hyperplasia was shown to be caused by a c-myc dependant hyperproliferation of acinar cells (Strom et al., 2007). Here again, the endocrine cells are unaffected by the artificial activation of the Wnt pathway.

In conclusion, during pancreas development Wnt signaling appears to have a bi- phasic effect. An active Wnt pathway has an inhibitory role early during pancreas specification, while it is also required later for proper pancreas growth and differentiation.

Retinoic acid signaling

Retinoic acid (RA) is the active, oxidized form of vitamin A (retinol) and plays essential roles in morphogenesis and organogenesis. It is synthesized from circulating retinol in a two-step reaction involving alcohol and aldehyde dehydrogenases. RA binds to dimers of retinoic acid receptors (RAR) and retinoid x receptors (RXR), two families of nuclear receptors which act as heterodimers to control the transcription of genes. Typical RA target genes contain a RA-responsiveness element (RARE) in their promoter region (Ross et al., 2000). In zebrafish, RA is critical for both pancreas and liver specification and development.

Zebrafish treated with a pan-RA receptor antagonist (BMS493) completely lack endocrine and exocrine markers. Furthermore, treatment with exogenous RA resulted in the anterior expansion of the liver and pancreas at the expense of more anterior derivatives. Interestingly, in zebrafish, RA is required before 9.5 hpf for pancreas specification, thus, blocking RA signaling at 12.5 hpf has no significant effect (Stafford and Prince, 2002). Similarly, mice knockout for the retinaldehyde

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dehydrogease 2 (Raldh2) lack dorsal Pdx1 expression and a dorsal pancreas.

Raldh2 is expressed in the dorsal pancreatic mesenchyme but is absent from the mesenchyme surrounding the ventral bud. Thus, in Raldh2 mutant mice, the ventral pancreas forms normally (Martin et al., 2005). Furthermore, mice expressing a dominant-negative (dn) version of a RAR under the control of the Pdx1 promoter lack both dorsal and ventral pancreas and die perinatally (Ostrom et al., 2008). RA appears to be essential for both dorsal and ventral pancreas development but through different mechanisms. In addition, RA was also shown to promote both endocrine and exocrine differentiation in vivo (Jiang et al., 2008; Ostrom et al., 2008).

The role of vascular structures and mesenchyme

Pancreatic growth is initiated precisely where the endodermal epithelium contacts the endothelium of major blood vessels. At E8.5, the dorsal aortae lie immediately dorsal to the endoderm as two endothelial tubes lateral to the notochord.

Experiments on pancreatic explants showed that isolated endoderm grown in culture alone, without dorsal aorta tissue, is not able to differentiate into exocrine or endocrine pancreatic cells and Pdx1 and Insulin expression are not observed at the cellular level. Conversely, when the isolated endoderm is co-cultured with dorsal aorta Pdx1 and Insulin expression is restored (Lammert et al., 2001).

Kinase insert domain protein receptor (kdr, flk1) encodes a receptor of the vascular endothelial growth factor (vegf). In Flk^-/- mice, the development of endothelial cells and thus of blood vessels is hindered. In these mice few Pdx1+ cells appear in the dorsal endoderm and further bud formation and differentiation was halted. In contrast, ventral pancreatic endodermal cells expressed Pdx1 and Ptf1a, forming a smaller bud than in controls, devoid of Glucagon-expressing cells (Yoshitomi and Zaret, 2004). These results show that dorsal and ventral pancreas development and differentiation require a tight interaction with vascular structures.

The mesenchyme surrounding both dorsal and ventral buds plays a pivotal role in the specification, differentiation and growth of pancreatic cells. The LIM/homeodomain transcription factor Isl1 is expressed in the mesenchyme surrounding the developing dorsal bud and later in all adult islet endocrine cells. The Isl1^-/- embryo is

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characterized by an almost complete absence of mesenchyme surrounding the dorsal bud epithelium and markedly reduced Pdx1 expression. On the contrary the ventral pancreatic mesenchyme and epithelium developed normally. In addition, glucagon cells were absent from both dorsal and ventral buds and exocrine cells developed only from the ventral bud (Ahlgren et al., 1997). Similarly Cdh2 (N- cadherin) is expressed in the mesenchyme surrounding both dorsal and ventral buds.

Mice lacking Cdh2 show a specific dorsal pancreas agenesis due to an impaired mesenchymal cell survival whereas the ventral bud is not affected. In Cdh2^-/- embryos the dorsal bud epithelium expresses Pdx1 but fails to grow and differentiate (Esni et al., 2001). Cardiac specific expression of Cdh2 in Cdh2^-/- mice restores heart and vascular function and rescues formation of the dorsal pancreas. This rescue is not due to specific contact between the endothelial cells and the epithelium but to circulating factors such as sphingosine-1-phosphate (Edsbagge et al., 2005).

A general assessment of these various KO and co-culture experiments leads to the proposal that dorsal bud development seems to rely more critically on signals coming from the mesenchyme and vascular structures, whereas the ventral bud appears to be more autonomous for its development.

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Maintenance and self-renewal of pancreatic progenitors.

Organ development requires a complex balance between cell differentiation and self- renewal of progenitors. This balance is required in order for the organ to reach its appropriate mature functional size. During development, cell differentiation occurs to generate a functional organ while an appropriate pool of mulitpotent progenitors must be kept to promote organ growth. To this aim several signaling pathways crosstalk to correctly pattern the organs.

Notch signaling

During development, Notch signaling regulates a variety of cell fate decisions in epithelial as well as non-epithelial tissue. Notch receptors are single-pass transmembrane proteins that are activated upon ligand binding. In mice, five ligands have been identified, three Delta-like (Dll1,3 and 4) and two Jagged (Jag1 and Jag2), see (Gordon et al., 2008) for a recent review. Activation triggers the proteolytic cleavage of the Notch intracellular domain (NICD) that will subsequently shuttle to the nucleus. It then interacts with the DNA-binding protein recombination signal binding protein for immunoglobulin kappa J region (Rbpj) to activate the expression of Notch target genes of the Hairy and Enhancer-of-split (Hes) family of genes (reviewed in (Fischer and Gessler, 2007)). Notch1 and Notch2 are expressed in the pancreatic epithelium whereas Notch3 and Notch4 are expressed in the mesenchyme and later in endothelial cells (Lammert et al., 2000).

Upon Notch pathway disruption, as in delta-like (Dll)^-/- or Rbpj^-/- mice, the pool of Hes1+ cells in pancreatic primordia is reduced and these are hypoplastic, with accelerated endocrine and exocrine differentiation (Apelqvist et al., 1999; Esni et al., 2001; Fujikura et al., 2006). Similarly, mice knockout for Hes1 displays a severe hypoplasia due to the premature differentiation of pancreatic precursors into glucagon-expressing cells (Jensen et al., 2000). On the other hand, when the Notch pathway is artificially induced in Pdx1- or Ptf1a-expressing pancreatic progenitors, it disrupts both endocrine and exocrine differentiation as well as organ branching (Esni et al., 2004; Hald et al., 2003; Murtaugh et al., 2003). Tight regulation of Notch

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pathway activation is required to ensure regulated expansion and differentiation of pancreatic progenitors. Surprisingly, the double activation of Notch1 and Notch2 receptors does not recapitulate the same phenotype, i.e. accelerated endocrine or exocrine differentiation. Rbpj appears thus to be required independently of Notch to regulate pancreatic progenitor maintenance and differentiation. In Notch1/2^-/- and Rbpj^-/- mice, ductal cells differentiate normally and express Cytokeratin-19 suggesting that functional Notch signaling is not required for appropriate ductal cell differentiation (Nakhai et al., 2008). In the developing pancreas, Notch signaling is not only required to regulate the balance between endocrine and exocrine progenitors but also play important roles during growth and branching.

Sox9

SRY-box containing (Sox) transcription factors are inhibitors of cell differentiation in the central nervous system (CNS). Several Sox genes are expressed in the developing and adult pancreas. Sox11 is expressed in the mesenchyme surrounding the developing pancreatic buds whereas Sox4 and Sox9 are expressed in the pancreatic epithelium (Lioubinski et al., 2003; Wilson et al., 2005). Sox9 is expressed in all Pdx1-expressing cells from E9 to E12.5, often in association with Hes1. Sox9 regulates the expression of Onecut1 and Foxa2 and its own expression (Lynn et al., 2007). At E15.5 it is rarely observed in Neurog3- expressing cells but lineage tracing experiments showed that Neurog3-expressing cells derive from cells having expressed Sox9 (Seymour et al., 2008). Later, Sox9 is absent from differentiated endocrine, ductal and exocrine cells. Accordingly, Sox9- expression is unaffected in Neurog3 mutant mice.

These data suggest that Sox9 plays a role in the maintenance of pancreatic progenitors prior to endocrine or exocrine differentiation. This was confirmed in mice with a pancreatic conditional mutation of Sox9. Mutant mice had a small pancreatic rudiment with no sign of differentiation whatsoever (Seymour et al., 2007).

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- 30 - Cpa1

In the developing pancreas Carboxypeptidase A1 (Cpa1) mRNA is detected as early as E9.5. At E11.5, Cpa1 protein is present in a scattered population of pancreatic epithelial cells right before the onset of pancreas branching. These Cpa1+ cells do not at this stage express any mature endocrine hormone marker but express Pdx1, Ptf1a, high levels of cMyc and proliferate 3-5 times faster than trunk epithelial cells (Zhou et al., 2007). From E12.5, Cpa1 gets restricted to the branching tips and this expression pattern persists through successive branching and growth of the pancreatic tree. In the adult, Cpa1 is restricted exclusively to acinar cells. Surprisingly lineage tracing analysis showed that Cpa1+ cells before E13.5 give rise to all three major cell types of the pancreas. In striking contrast, Cpa1+ cells labeled from E13.5 give rise to acinar cell exclusively (Zhou et al., 2007). It has thus been proposed that multipotent Cpa1+ cells divide asymmetrically at the onset of branching morphogenesis, thereby propelling the multipotent pool of cells away from the primary axis, creating new branches (Figure 7) (Zhou et al., 2007).

Figure 7 Pancreas branching morphogenesis is directed by a pool of proliferating Cpa1- expressing multipotent progenitors (purple). Upon proliferation, Cpa1-expressing cells generate the trunks that harbour ductal and endocrine progenitors (blue and green cells).

Modified from (Zhou et al., 2007)

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- 31 - Specification of endocrine progenitors

During pancreas development, there are two temporal waves of endocrine differentiation defined as the “first” and “secondary transitions” (Pictet et al., 1972).

The first transition takes place between E9.5 and approximately E12.5. During this period, individual early hormone-expressing cells differentiate and most of them express Glucagon, sometimes in combination with Insulin or PP (Herrera et al., 1991;

Upchurch et al., 1994). The secondary transition, which initiates around E12.5, gives rise to fully mature, single hormone expressing cells and the majority of newborn islet cells are formed during this period.

Lineage tracing experiments have shown that hormone-expressing cells do not transdifferentiate over time. Glucagon- and Insulin- expressing cells contribute only to their respective adult cell types, ruling out a contribution of double insulin/glucagon positive cells to the formation of adult endocrine cells (Herrera, 2000; Herrera et al., 1998). Interestingly, when early neurog3-expressing endocrine precursors are labeled around E8.5 their progeny can still be observed in adult islets (Gu et al., 2002). The precise role and fate of the early insulin+/glucagon+ double positive cells is still controversial and whether these early endocrine cell contribute to the formation of adult islets is still a matter of debate.

The specification of endocrine progenitors and subsequently of differentiated endocrine cells is still unclear, even though several key players have already been identified.

Prox1

Prospero-related homeobox (Prox1) is expressed in both the liver and pancreatic buds at E9.5 (Burke and Oliver, 2002). In the E13.5 pancreatic epithelium, Prox1 is expressed along with Pdx1 in all epithelial cells with the exception of some epithelial cells expressing only Prox1. Prox1 is also expressed in all Neurog3- expressing endocrine progenitors. At E15.5, Prox1 gets restricted to Isl1+ endocrine cells but is almost absent from differentiating exocrine cells. Interestingly this

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expression pattern changes after birth where Prox1 is expressed in all but exocrine cells although at low levels in islet β-cells (Wang et al., 2005).

At E11.5, mutant mice for Prox1 have a smaller pancreas that fails to branch by E15.

During the secondary transition, Prox1^-/- pancreas show a reduced number of Neurog3+ endocrine precursors and individual Isl1+ endocrine cells (Wang et al., 2005). Conversely, between E13.5 and E14.4, the number of exocrine progenitors expressing Ptf1a is substantially increased which results in an increased number of differentiated exocrine cells expressing Cpa1 or Amylase (Wang et al., 2005). Prox1 seems to be important for regulating the balance between endocrine and exocrine progenitors during pancreatic development.

Neurogenin 3

Neurog3 is a basic loop helix (bHLH) transcription factor expressed in a subset of Pdx1+ cells of the developing pancreas. Its induction appears to be negatively regulated by Hes1 via Notch-mediated signaling (Apelqvist et al., 1999; Lee et al., 2001). Neurog3 expression takes place in two temporal waves, concomitant to the first and secondary transitions: from E8.5 to E11 and from E12 to birth with a peak of Neurog3-expressing cells at E15.5 (Villasenor et al., 2008).

In the developing pancreas, its expression at the single cell level is transient and short as Neurog3 is never co-expressed with hormones or other markers of differentiated cells. Lineage tracing studies showed that Neurog3-expressing cells give rise to all adult islet cells (Gu et al., 2002; Herrera et al., 2002). Additionally, mice mutant for Neurog3 specifically lack endocrine cells, making Neurog3 the earliest known specific endocrine precursor cell marker (Gradwohl et al., 2000;

Schwitzgebel et al., 2000).

Neurog3 is both necessary and sufficient to reprogram a pancreatic cell towards an endocrine fate. Overexpression of Neurog3 in human or mouse pancreatic duct cells was sufficient to induce the endocrine differentiation program (Gasa et al., 2004;

Heremans et al., 2002). However, a precise temporal expression of Neurog3 is required for to generate adequate proportions of the different endocrine cell types.

Early ectopic expression of Neurog3 in Pdx1-expressing cells results in a precocious

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differentiation of pancreatic progenitors into small clusters of glucagon-positive cells (Apelqvist et al., 1999; Johansson et al., 2007; Schwitzgebel et al., 2000). Its conditional expression later in the same cells gives rise to PP-, Insulin- and Somatostatin-expressing cells in chronological orders (Johansson et al., 2007). It thus seems that there is a temporal window of competence for each endocrine cell type during pancreas development. The subsequent endocrine differentiation program, once Neurog3 expression has ceased, relies on downstream activated factors.

Whether Neurog3-expressing cells still persist after birth is still a matter of debate.

The activity of the Neurog3 promoter has been reported in adult pancreas (Gu et al., 2002) but the protein has never been observed in healthy adult mice. However, after pancreatic duct ligation (PDL), a surgical procedure that triggers pancreatitis and an important tissular remodeling of the pancreas, the protein is re-expressed in adult ducts (Xu et al., 2008).

Neurod1

Neurod1 is a bHLH transcription factor expressed early in the developing pancreas with a pattern similar to Neurog3. At E9, a subset of epithelial cells expresses Neurod1 and most of these cells co-express glucagon. Later, Neurod1 is expressed in all endocrine cells of the developing and adult pancreas indicating that, unlike Neurog3, Neurod1 expression is maintained in differentiated endocrine cells (Naya et al., 1997). Neurog3 activates directly Neurod1 expression which in turn triggers cell cycle exit of the endocrine precursor cells (Huang et al., 2000).

Subsequently, Neurod1 binds to the E box sequences localized on the promoters of Insulin and Glucagon and regulates their expression (Dumonteil et al., 1998; Naya et al., 1995). Mice knockout for Neurod1 die after birth of severe diabetes. They have a 60% reduction of endocrine cells and fail to develop morphologically distinct islets.

Newborn mutant pancreas have 41%, 37% and 22% of α-, β- and δ-cells instead of the 80%, 15% and 5% expected (Naya et al., 1997). The phenotype of NeuroD1^-/- mice varies with different genetic backgrounds. Mutant mice highly enriched in 129/SvJ background survive several months due to a significant islet cell neogenesis after birth (Huang et al., 2002).

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NeuroD1 is thus required early to regulate cell cycle exit and hence, allow the differentiation of endocrine progenitors. Later NeuroD1 controls endocrine gene expression and identity

Insm1 (Insulinoma-associated 1)

Insm1 is a zinc-finger transcription factor first identified in human β-cell insulinomas (Goto et al., 1992). In mice it was identified in ductal cells transduced with an adenovirus expressing Neurog3. Furthermore, it was shown that Neurog3 can bind to and activate the Insm1 promoter. At E10.5 its expression pattern is similar to Neurog3. The number of Insm1-expressing cells peaks around E15.5 and decreases below detection limits from E18.5 on as shown by in-situ hybridization (ISH). During pancreas development, Insm1+ cells rarely coexpress hormones, suggesting that Insm1 is expressed after Neurog3 but before NeuroD1. Furthermore, Neurog3^-/- mice fail to express Insm1 but, Insm1 expression is normal in NeuroD1^-/- mice (Mellitzer et al., 2006).

In the adult, the Insm1 expression pattern is debated. Although it is not detected by ISH, mice with a LacZ reporter knocked into the Insm1 coding sequence show a strong β-gal expression in all endocrine cells of adult islet (Gierl et al., 2006). Insm1^-/- mice have normal α-cell numbers but show a strong reduction in β- and δ-cells and an significant increase in PP-cells. An Insm1 binding site has been identified on the Insulin promoter and Insm1 appears to negatively regulate Insulin transcription. This repression is mediated by the recruitment of both cyclin D1 and histone deacetylase 3 (HDAC-3) (Wang et al., 2008a). Insm1 appears therefore to regulate islet cell fate allocation by promoting β- and δ-cell differentiation while preventing PP-cell differentiation.

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- 35 - Nkx2-2

NK homeodomain transcription factors are named for the presence of an amino-terminal NK decapeptide (the function of which is unknown) and are key regulators of development and differentiation in several tissues.

During pancreas development, Nkx2-2 is first expressed in the whole epithelium from E9.5 to E12.5. Then, as the pancreatic program resolve into endocrine and exocrine cells, Nkx2-2 expression persists only in the endocrine cells. In the adult pancreas, Nkx2-2 is expressed in α-, β- and PP-cells but not in δ-cells (Sussel et al., 1998).

Thus, it appears that Nkx2-2 is expressed at three critical stages of islet cell differentiation: first, early in the whole pancreatic epithelium, then in a subset of Neurog3-expressing cells and finally in the majority of differentiated islet cells.

Interestingly, Nkx2-2 has three variable first exons (exons 1a, 1b and 1c). It has been shown that 1b is expressed in islet precursor cells and 1a in differentiated islet cells, but 1c has not been detected (Watada et al., 2003). Mice mutant for Nkx2-2 completely lack β-cells, have a strongly reduced number of α-cells but the PP-cell number is only slightly altered and δ-cells are unaffected. Unfortunately, the absolute number of endocrine cells has not been addressed (Sussel et al., 1998). In mutant islets, a large population of endocrine cells expresses Synaptophysin and Chromogranin A but none of the four islet hormones. They were identified as a fifth type of endocrine cells (ε-cells), expressing ghrelin which expands massively in Nkx2-2 mutant islets (Prado et al., 2004).

In conclusion, Nkx2-2 is required for the specification of β- and α-cells and appears to repress the differentiation of ε-cells or active a diversion process.

Nkx6-1 and Nkx6-2

Like Nkx2-2, Nkx6-1 is a homeodomain transcription factor, but it falls in a divergent subfamily within the NK homeodomain family (Rudnick et al., 1994). Nkx6-1 expression pattern is similar to that of Nkx2-2. At E10, it is expressed in pancreatic progenitor cells in combination with Pdx1 and Ptf1a (Hald et al., 2008; Jensen et al., 1996). Interestingly, Insulin+/Glucagon+ early hormone expressing cells do not express either Nkx6-1 or Pdx1 (Oster et al., 1998). At the beginning of the secondary

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transition, Nkx6-1 is expressed in ductal and periductal cells expressing Pdx1 or Neurog3 and in Insulin-expressing cells. At E15.5, all Nkx6-1 expressing cells also express Nkx2-2, but many Nkx2-2 expressing cells do not express Nkx6-1. Finally, at later developmental stages and postnatally, Nkx6-1-expression is restricted to β-cells (Sander et al., 2000). As expected, the disruption of Nkx6-1 impairs the generation of 98% of β-cells, sparing those coming from the first transition. Interestingly the phenotype of Nkx2-2 and Nkx6-1 compound mutant is similar to that of Nkx2-2, indicating that Nkx6-1 acts downstream of Nkx2-2 (Sander et al., 2000).

By using transgenic mice in which the Nkx6-2 coding sequence was replaced by a Tau-lacZ cassette, Henseleit and colleagues showed that Nkx6-2 is coexpressed with Nkx6-1 in the early undifferentiated epithelium. At E13.5 the majority of NK- expressing cells express only Nkx6-1 and at E15.5, Nkx6-1 and Nkx6-2 are found in distinct domains. Nkx6-2 never colocalizes with insulin or Neurog3 but is expressed with glucagon and amylase. Using the same approach, they also showed that Nkx6-2 is expressed in Pdx1-expressing progenitors giving rise to both Neurog3- and Insulin- expressing cells. Finally, around birth, Nkx6-2 disappears from the pancreas (Henseleit et al., 2005).

Initially, at E10.5, Nkx6-2 expression is not affected in Nkx6-1 knockout mice but it is later upregulated at E13.5 and E15.5. However, Nkx6-1 expression is not affected in Nkx6-2 mutants indicating that Nkx6-1 represses Nkx6-2 but not conversely. Nkx6-2 mutants are identical to controls indicating that Nkx6-1 can compensate for the loss of Nkx6-2. More interesting, a transgene driving the expression of Nkx6-1 under the Neurog3 promoter in Nkx6-1 mutant mice is unable to specify β-cells whereas its transgenic expression in Pdx1-expressing cells can. Similarly, a Pdx1-promoter driven Nkx6-2 transgene has the same ability to rescue β-cell specification in Nkx6-1 mutants (Nelson et al., 2007). It indicates that both Nkx6-1 and Nkx6-2 have the potential to specify β-cells in Pdx1-expressing pancreatic progenitors. However in Nkx6-1^-/- mice, Nkx6-2 is not induced in Pdx1-expressing pancreatic progenitors and β-cells specification is impaired.

It indicates that Nkx6-1 and Nkx6-2 have partially overlapping function but Nkx6-1 is not dependant on Nkx6-2 for β-cell specification whereas Nkx6-2 cannot specify β- cell in the absence of Nkx6.1.

Analysis of the differentiation potential of murine pancreatic islet precursor cells: an In Vivo clonal analysis

Thesis

Reference

Analysis of the differentiation potential of murine pancreatic islet precursor cells: an In Vivo clonal analysis

Analysis of the Differentiation Potential of Murine

Pancreatic Islet Precursor Cells: an In Vivo Clonal Analysis

THÈSE

Table of Contents

Acknowledgments

I Résumé en français

II English Summary

III Introduction