FGFs and Wnts in pancreatic growth and

β-cell function

Stella Papadopoulou

Umeå Centre for Molecular Medicine (UCMM) Umeå University

Umeå, Sweden, 2005

Front cover: Wholemount immunostaining of adult mouse pancreas with antibodies against α- smooth muscle actin (green) and insulin (red)

Copyright 2005 Stella Papadopoulou ISBN 91-7305-866-1

Printed by Solfjädern Offset AB Umeå, Sweden, 2005

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As you set out for Ithaca pray that the journey is long, full of adventure, full of discovery. Laistrygonians, Cyclops, angry Poseidon – do not be afraid of them: you’ll never find things like that on your way as long as you keep your thoughts raised high, as long as a rare excitement stirs your spirit and your body. Laistrygonians, Cyclops, wild Poseidon – you won’t encounter them unless you bring them deep inside your soul, unless your soul sets them up in front of you.

Then pray that the journey is long. That the summer mornings are many, that you will enter ports seen for the first time with such pleasure, with such joy! Stop at Phoenician markets and purchase fine merchandise, mother-of-pearl and corals, amber and ebony, and pleasurable perfumes of all kinds, buy as many pleasurable perfumes as you can; visit hosts of Egyptian cities, to learn and learn from those who have knowledge. Always keep Ithaca fixed in your mind. To arrive there is your ultimate goal. But do not hurry the voyage at all. It is better to let it last for long years; and even to anchor at the isle when you are old, rich with all you have gained on the way, not expecting that Ithaca will offer you riches.

Ithaca has given you a beautiful voyage. Without her you would never have taken the road. But she has nothing to give you now.

And if you have found her poor, Ithaca has not defrauded you. With such great wisdom you have gained, with so much experience you must surely have understood by then what Ithacas mean.

Constantinos P. Kavafis, Ithaca (1911)

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TABLE OF CONTENTS

1. ABSTRACT 7

2. PAPERS IN THIS THESIS 9

3. INTRODUCTION-PANCREAS DEVELOPMENT:

Past and Present 11

3.1 What is all the fuss about? 11

3.2 The mature pancreas 12

3.3 Endoderm development 13

3.4 Early pancreatic development and patterning 14

3.5 Growth of the pancreatic primordium 18

3.6 Specification and differentiation of pancreatic

cell types 19

3.6.1 Specification of endocrine and exocrine cell fates by Notch

signalling 21

3.6.2 Factors involved in endocrine cell differentiation and

specification 23

3.6.3 Factors involved in exocrine cell differentiation and

specification 27

4. AIMS OF THIS THESIS 29

5. RESULTS AND DISCUSSION 30

5.1 PART 1-FGF SIGNALLING IN PANCREATIC GROWTH

AND HOMEOSTASIS 30

5.1.1 Background 30

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5.1.2 More is Less: FGF in pancreatic epithelial cell proliferation 35

5.1.3 Enough is Enough: Persistent FGF signalling in β-cell leads to

diabetes in mice 45

5.2 PART 2- WNT SIGNALLING IN PANCREATIC GROWTH

AND HOMEOSTASIS 53

5.2.1 Background 53

5.2.2 Less can be lesser: Wnt signalling in pancreatic growth and

function 58

6. EPILOGUE-Pancreatic development and prospects for

therapy 68

7. CONCLUSIONS 71

8. ACKNOWLEDGEMENTS 73

9. REFERENCES 75

10.PAPERS I-III 89

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1.ABSTRACT

The pancreas is an endodermally derived organ that initially appears as a dorsal and ventral protrusion of the primitive gut epithelium. Mesenchymal-epithelial interactions are pivotal for proper pancreatic growth and development. The pancreatic progenitor cells present in the early pancreatic anlagen proliferate and eventually give rise to all pancreatic cell types. The Fibroblast Growth Factor 2b (FGFR2b) high-affinity ligand Fibroblast Growth Factor 10 (FGF10) has been linked to pancreatic epithelial cell proliferation and we have previously shown that Notch signalling controls pancreatic cell differentiation via lateral inhibition. By overexpressing FGF10 under the control of the Ipf1/Pdx1 promoter in mice, we have shown that persistent FGF10 activation in the embryonic pancreas of transgenic mice perturbs pancreatic epithelial cell proliferation and also inhibits pancreatic cell differentiation by maintaining Notch activation. In the Ipf1/Fgf10 transgenic mice, the pancreatic epithelial cells are ‘locked’ in an undifferentiated progenitor-like state with sustained proliferative capacity. Collectively, our data suggest a key role for FGFR2b/FGF10 signalling in the regulation of pancreatic growth and differentiation and that FGFR2b/FGF10 signalling interact with the Notch signalling pathway.

Glucose homeostasis in mammals is critically dependent on co-ordinated glucose uptake, oxidative metabolism and insulin secretion in β-cells. Although, several key genes controlling various aspects of glucose sensing, glucose metabolism, insulin expression and secretion have been identified, we know relatively little about the molecular mechanisms that induce and maintain the expression of genes required for glucose-stimulated insulin secretion (GSIS) in β-cells. Attenuation of FGFR1c signalling leads to diabetes in mice. Overexpression of FGF2, a high-affinity FGFR1c ligand, under the control of the Ipf1/Pdx1 promoter also leads to diabetes in mice. The Ipf1/Fgf2 mice present with normal endocrine and exocrine differentiation but display impaired glucose-stimulated insulin secretion (GSIS), perturbed expression of genes required for glucose sensing uptake together with oxidative metabolism and increased expression of the FGF-signalling inhibitors Spry-2 and Pyst1/MKP3 in β-cells. Thus, stringent control of FGF signalling activation appears crucial for the maintenance of the regulatory circuit that ensures proper GSIS in pancreatic β-cells and hence normoglycaemia.

The Wnt family of ligands via their receptors Frizzled (Frz) have been shown to mediate mesenchymal-epithelial interactions and cell proliferation in a variety of different systems. Expression of a plethora of Wnt ligands and Frz receptors has been previously reported in the pancreas and mice missexpressing Wnt1 and Wnt5a under the Ipf1/Pdx1 promoter display severely perturbed development. Here, we show the temporal and spatial expression of Wnt4, Wnt7b and Frz3 at different stages of pancreas development. Onset of Wnt4 expression is observed in a subset of ngn3+ cells at e13 and strong expression is detected at late stages predominantly in differentiated α-cells. Wnt7b expression is detected in the ductal epithelium between e13 and e15. Frz3, like FGFR2, is expressed in the pancreatic epithelium during the proliferative phase of the embryonic pancreas in mice, i.e

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from e10-e15 implicating a potential role for this receptor in pancreatic epithelial cell proliferation. To elucidate the role of Wnt signalling in the pancreas, we overexpressed a dominant negative form of mouse Frz8 under the Ipf1/Pdx1 promoter in mice. The Ipf1/Frz8CRD mice display severe pancreatic hypoplasia demonstrating that attenuation of Wnt signalling in the pancreas leads to perturbed pancreatic growth. Nevertheless, the transgenic mice present with normal endocrine and exocrine differentiation and remain normoglycaemic. The maintenance of normoglycaemia in these mice appears to be the consequence of a relative increase in endocrine cell number per pancreatic area combined with enhanced insulin biosynthesis and insulin secretion. Collectively our data provide evidence that Wnt signalling is required pancreatic growth but not adult β-cell function.

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2. PAPERS IN THIS THESIS This thesis is based on the following papers that will be referred to in the

text by their corresponding Roman numerals (I-III).

I. Hart AW∗, Papadopoulou S∗ and Edlund H (2003) Fgf10 Maintains

Notch Activation, Stimulates Proliferation, and Blocks Differentiation

of Pancreatic Epithelial Cells. Dev. Dyn 228: 185-193

II. Papadopoulou S and Edlund H (2005) Persistent FGF signalling in

β-cells leads to diabetes in mice. Manuscript

III. Papadopoulou S and Edlund H (2005) Attenuated Wnt signalling

perturbs pancreatic growth but not pancreatic function. Submitted

∗The first two authors contributed equally to this work

Article I is reproduced with permission from the respective copyright holder.

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3. INTRODUCTION PANCREAS DEVELOPMENT: Past and Present

3.1 What is all the fuss about?

Had it not been for its pathology, it is unlikely that developmental biologists

would ever be keen to study the pancreas as a model system. It arises

early in development and it is small and barely accessible. When it is big

enough to become accessible, its branching morphogenesis is so fine and

atypical that you begin to wonder whether you chose the right profession!

And if those reasons weren’t good enough, try this one: the differentiating

pancreas is virtually bubbling with proteases and RNAases making genetic

analysis a one-way trip to Elm Street!

Yet, ignoring the medical importance of the pancreas is a luxury we simply

can’t afford. And the figures speak for themselves. The World Health

Organization estimated in 1997 that 143 million people were afflicted with

diabetes worldwide and predicted that this figure is likely to double by 2025,

making diabetes one of the most debilitating diseases of our time! Even

more so, in the developed world, diabetes surprises us with a goody bag

full of secondary complications such as retinopathy, nephropathy,

neuropathy, fatty liver, liver cancer and cardiovascular disease.

Having said that, scientists today are faced with the daunting task of

unraveling new and more efficient ways for prevention, treatment or even

cure for the disease. For this purpose, research with emphasis on the

endocrine pancreas has grown dramatically and a plethora of discoveries

regarding pancreas development has emerged over the past ten years or

so. The efforts undertaken by developmental biologists in the field today

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diverge towards a common consensus: to identify and delineate the

molecular cues that govern pancreas development and function.

3.2 The mature pancreas (Figure 1)

The principal function undertaken by the pancreas is glucose homeostasis,

a key aspect of which is the constant monitoring of blood glucose levels.

The mature pancreas has morphologically and functionally distinct

endocrine and exocrine components (Figure 1).

Figure 1. The adult pancreas A) anatomy of the adult pancreas B) the exocrine pancreas consists of duct and acinar cells C) the endocrine pancreas in organized into islets of Langerhans that contain 4 different types of endocrine cells each producing a distinct peptide (illustration by Ulf Ahlgren)

The exocrine part comprises some 95-99% of the pancreas and includes

the acinar and ductal cells. The acinar pancreas produces digestive

enzymes, such as trypsin and amylase, which are shunted into the gut via

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the duct cells to aid digestion and absorption. Digestive enzymes are

secreted into the acinar lumens, which drain into small ducts. Like tree

branches, the ducts merge and feed into progressively larger structures

that eventually connect with the common bile duct.

Mature endocrine cells are organised into endoderm-derived globular

clusters of cells dispersed throughout the exocrine tissue called islets of

Langerhans. Islets contain four principal cell types that are present in

varying proportions: the insulin-producing β-cells that lie in the core of the

islet (60-80%), the glucagon-producing α-cells (15-20%), somatostatin-

producing δ-cells and pancreatic polypeptide-producing PP-cells that are all

found in the periphery. The islets make up only about 1-2% of the entire

organ.

But how does the adult pancreas develop into this elaborate sensory

apparatus and what is the molecular milieu that makes all this possible?

3.3 Endoderm development

Early in development, an embryo becomes oriented from top-to-bottom

(anterior-posterior; A-P) and from front-to-back (dorsal-ventral; D-V). Later

on, via a process called gastrulation, cells are partitioned into 3 categories

forming 3 distinct layers: the ectoderm, which gives rise to the skin and the

central nervous system; the mesoderm that forms blood, bones and

muscle; and the endoderm from where the respiratory and digestive tracts

develop. After gastrulation, the endoderm is shaped as a one-cell layer

thick sheet of approximately 500 cells (in mice) that will form the epithelial

lining of the esophagus, lungs, stomach, intestines as well as being the

major component of many glands including the thyroid, thymus, pancreas

and liver. Ensuing morphogenetic movements push the endodermal sheet

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towards the inside of the embryo eventually forming a primitive tube. This

tube later becomes the gut tube and evaginations (buds) from the gut grow

and branch into differentiated fully-functional organs that control functions

such as taste, gas-exchange, digestion, nutrient absorption, glucose

homeostasis, detoxification, blood clotting and hematopoiesis.

3.4 Early pancreatic development and patterning

Classical embryological experiments performed in the 1960s have shed

light onto fundamental aspects of pancreas development. The pancreas

derives from the upper, duodenal part of the primitive gut via a direct dorsal

and ventral evagination of the epithelium posterior to the developing

stomach. In mice, the early pancreatic buds become morphologically visible

at embryonic day 9 (e9) in mice. The region of the primitive gut where the

pancreas will later form becomes, however, committed to a pancreatic fate

already at e8.5 (Wessells and Cohen, 1967). Prior and during budding, the

pancreatic primordium expresses the homeodomain protein IPF1 (Ohlsson

et al, 1993; Ahlgren et al, 1996; also known as PDX-1, IDX-1, STF-1;

Leonard et al, 1993; Miller et al, 1994). All pancreatic types derive from

IPF1/PDX1+ progenitors (Ohlsson et al, 1993; Gu et al, 2004). In the

absence of a functional Ipf1/Pdx1 allele pancreatic development arrests

after budding (Jonsson et al, 1994; Ahlgren et al, 1996; Offield et al, 1996)

providing evidence that Ipf1/Pdx1 is not critical for the induction of the

pancreatic program but for development beyond bud formation.

Furthermore, studies by Golosow and Grobstein along with these by Haffen

and associates have shown that interactions between the gut epithelium

and the surrounding mesenchyme are essential in ensuring proper

development of the gut and its associated organs, including the pancreas

(Golosow and Grobstein, 1962; Haffen et al, 1987). Evidence from in vitro

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culture experiments and transgenic mouse models have shown that proper

differentiation of the pancreatic mesenchyme, which in turn ensures proper

pancreatic epithelial development, is dependent upon excluding members

of the Hedgehog family of signalling molecules, namely sonic hedgehog

(shh) and indian hedgehog (ihh), from the presumptive pancreatic region of

the gut. Both shh and ihh are uniformly expressed in the endoderm anterior

and posterior to the pancreas but no expression of either shh or ihh is

detected in the dorsal and ventral pancreatic epithelium (Bitgood and

McMahon, 1995; Apelqvist et al, 1997). In mice with ectopic expression of

shh under the control of the Ipf1/Pdx1 promoter the pancreatic

mesenchyme differentiates into intestinal smooth muscle and the

pancreatic epithelium adopts an intestinal fate but is still able to generate a

few pancreatic cell types (Apelqvist et al, 1997).

Complementary studies by Melton and associates where early chick

endodermal rudiments were in vitro cultured together with chick notochord

explants, demonstrated a role for the notochord in suppressing shh

expression in committed pancreatic epithelium and identified signalling

factors activinβB and FGF2, both expressed early in the notochord, to be

key factors in this notochord-mediated repression of shh in the pancreatic

anlagen (Hebrok et al, 1997; Kim et al, 1998). Moreover, while it is clear

that the notochord is involved in excluding shh from the dorsal pancreatic

endoderm, a similar effect on the ventral pancreatic endoderm is dubious

due to the fact that the notochord does not lie in direct contact with the

ventral gut epithelium and also because it is not currently known whether

the latter initially expresses shh and ihh. Hence, signals repressing or

allowing expression of these proteins in the ventral gut epithelium must

derive from other tissues such as the adjacent cardiogenic mesoderm and

septum transversum.

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Indeed, the cardiac mesoderm controls ventral pancreas specification via

secretion of FGFs. Specification of the ventral pancreas occurs at the same

time as that of the liver in the same domain of precursor cells (Deutsch et

al, 2001). Mouse embryonic explant experiments have suggested that the

default state of the ventral foregut endoderm is the activation of the

pancreatic program. FGFs from the cardiac mesoderm determine the fate

of this bipotential cell population such that when present, they induce the

expression of shh which, in turn, turns off the ventral pancreas specification

program thereby allowing for the primitive liver bud to appear. In the

absence of heart mesoderm-derived FGFs, shh expression is prevented

and the pancreatic program proceeds undisturbed (Deutsch et al, 2001).

The Hlxb9 gene, which encodes the homeobox transcription factor Hb9, is

transiently expressed in the early pancreatic buds and in the dorsal bud its

expression precedes that of IPF1/PDX1 (Harrison et al, 1999, Li et al,

1999). Hb9 is also expressed in the notochord. Due to the spatial alignment

along the AP-axis, it seems likely that the notochord may help establish

Hlxb9 expression in the dorsal endoderm, which in turn permits pancreatic

development to occur. In mice lacking Hlxb9, IPF1/PDX1 is not expressed

and the dorsal pancreas is entirely absent whereas the ventral pancreas

develops normally until later stages which may reflect intrinsic differences

between the dorsal and ventral buds. Conversely, mice lacking the

Ptf1a/p48 bHLH transcription factor exhibit normal bud formation, whereas

the ventral pancreas not only fails to bud, but becomes integrated into the

surrounding duodenum (Kawaguchi et al, 2002). This was shown by

knocking in Cre recombinase to the Ptf1a/p48 locus disrupting the gene

and generating a line-tracing reagent simultaneously. In the heterozygous

state (Ptf1a Cre/+), Cre-catalysed activation of LacZ reporter showed that

Ptf1a+ cells contribute to all cell types of the dorsal and ventral pancreas

but not to the adjoining duodenum. However, in the homozygous state

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(Ptf1a Cre/Cre) LacZ expression ventrally is observed in the duodenal wall,

apparently contributing to normal intestinal epithelium.

Taken together, these data suggest that extrinsic factors regulate A-P

patterning of the endoderm to establish the pancreatic domain. This

positional information is interpreted by transcription factors that specify

pancreatic fate, promote IPF1/PDX1 expression and epithelial budding

(Figure 2).

Figure 2. Schematic representation of early pancreatic development with emphasis on the extrinsic factors involved in the induction and specification of the pancreatic program. In the mouse, the notochord contacts the dorsal pancreatic endoderm and the ventral spinal chord until ∼14 somite stage. Notochord-derived factors repress Shh expression at the prospective pancreatic domain allowing the induction of the dorsal pancreatic program. At 20 somites, the dorsal aorta lies between the gut endoderm and the notochord sending additional extrinsic signal required for pancreas development (Lammert et al, 2001). At 25 somites, mesenchyme has accumulated around the dorsal pancreatic bud and at ∼28 somites (e9), IPF1/PDX1 expression can be detected in the early pancreatic progenitor cells. All drawings correspond to transverse views of the dorsal pancreatic anlage at the somite stages listed in the figure. (illustration by Ulf Ahlgren/A. Sandström).

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3.5 Growth of the pancreatic primordium

Following budding, the two distinct pancreatic primordia begin to grow,

branch and eventually fuse into a single bipolar organ. The developing

epithelium is surrounded by the mesenchyme and classic culture

experiments performed in the 60’s comparing in vitro pancreatic epithelial

cell proliferation in the absence of presence of mesenchyme highlighted the

importance of the mesenchyme for growth and (exocrine) differentiation

(Golosow and Grobstein, 1962, Wessells and Cohen, 1967). Further

evidence supporting the importance of mesenchyme in pancreas

development derived from the phenotype of the Isl1-/- mice. The LIM

homeodomain transcription factor ISL1 is expressed in the dorsal mesenchyme during bud formation. In Isl1-/-embryos the dorsal pancreatic

mesenchyme is missing and dorsal IPF1/PDX1 expression is reduced

(Ahlgren et al, 1996). Moreover, the dorsal bud of the Isl1-/- embryos fails

to undergo exocrine differentiation in vitro, which usually correlates with

growth. However, co-culture with wild-type mesenchyme rescues dorsal

exocrine differentiation demonstrating the absolute requirement of the

mesenchyme for proper growth and differentiation. Similar phenotypes are

observed in mice lacking N-cadherin, also expressed in the mesenchyme

(Esni et al, 2001) and in mice lacking the homeobox gene Pbx1, albeit less

severe (Kim et al, 2002). In both cases, exocrine differentiation is again

rescued by co-culture with wild-type mesenchyme. The Pbx1 mutants

exhibit severe hypoplasia of the dorsal pancreas as well perturbed acinar

development (Kim et al, 2002). Interestingly, Pbx1 forms DNA-binding

complexes with IPF1/PDX1 (Asahara et al, 1999) and transgenic mice that

express a mutant form of Pbx1 that is unable to bind to IPF1/PDX1, only

partially rescue the growth when crossed into a Ipf1/Pdx1 null background

(Dutta et al, 2001). These data suggest a role of IPF1/PDX1 in epithelial

cell proliferation that is mediated partly through its interaction with Pbx1.

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A number of cell-extrinsic mesenchymal factors are shown to be required

for pancreatic growth. One recently identified mesenchymal signal is

fibroblast growth factor 10 (FGF10). Mice lacking FGF10 display dorsal and

ventral pancreatic hypoplasia, apparently as a result of decreased epithelial

proliferation (Bhushan et al, 2001). Other mesenchymal signals that

promote growth of the pancreatic epithelium include members of the

epidermal growth factor family (EGF) as EGF receptor mutants show

moderately impaired pancreatic growth (Miettinen et al, 2000) and EGF

also promotes epithelial proliferation in vitro (Cras-Maneur et al, 2001). In

both cases the hypoplasia observed in the mutant mice is coupled with

decreased IPF1/PDX1 expression. To better define the requirement for

IPF1/PDX1 during pancreatic growth, Holland and co-workers replaced the

Ipf1/pdx1 coding region with tetracycline transactivator protein (tTA) to

define the requirement for IPF1/PDX1 in pancreas growth. When combined

with a Ipf1/Pdx1 transgene under Tet regulation, expression of IPF1/PDX1

could be temporally controlled in compound mutant embryos. Hence, by

downregulation of IPF1/PDX1 after bud outgrowth, Holland and associates

showed that IPF1/PDX1 is continuously required for epithelial cell

proliferation in the pancreas (Holland et al, 2002). Finally, the phenotype

displayed by Ptf1a/p48 mutant mice support a role also for this transcription

factor in pancreatic epithelial cell proliferation. The dorsal bud of Ptf1a/p48

mice is specified normally but its outgrowth and branching morphogenesis

are considerably reduced compared to control buds (Kawaguchi et al,

2002).

3.6 Specification and differentiation of pancreatic cell types

By e9.5 scattered glucagon producing cells are detected by

immunohistochemistry marking the onset of endocrine cell differentiation.

Organisation of exocrine cells into acini and ducts takes place first at

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around e14.5. At this point, endocrine cells are embedded as individual

cells in ducts or in small clusters (“endocrine streaks”) distinct from the

ducts. In mice, formation of islets with the stereotyped architecture with β-

cells in the centre of the islet surrounded by non β-cells in the periphery, is

evident first at e18.

The appearance of glucagon-expressing cells within the primitive buds at

e9.5 is the first evidence of cytodifferentiation of pancreatic cells. It was

therefore suggested that these early glucagon-expressing cells are

precursors of all cells of the endocrine cell lineage. However, lineage

tracing experiments conducted by Herrera and coworkers proved this

notion to be wrong and showed that endocrine cells types develop

independently from non-hormone-producing IPF1/PDX1-expressing

precursor cells (Herrera, 2000; Herrera et al, 1994). Furthermore, studies

by Gu and coworkers showed that all endodermally derived cells of the

pancreas - both endocrine and exocrine- originate from IPF1/PDX1-

expressing progenitor cells (Gu et al, 2004). Furthermore, modified

knockout approaches by Kawaguchi and associates revealed that nearly all

pancreatic cell types can be traced back to Ptf1a-expressing progenitor

cells showing that Ptf1a, like IPF1/PDX1, is expressed in undifferentiated

pancreatic precursors (Kawaguchi et al, 2002). Together with E12 and

HEB/REB/Alf1, Ptf1α is a transcriptional complex of the bHLH transcription

factor p48 and binds to a conserved element of the regulatory region of

exocrine genes. The presence of cells multipotential cells that co-express

IPF1/PDX1 and Ptf1α/p48 was confirmed with combined DNA microarray

analysis (Gu et al, 2004) and single-cell PCR by Chiang and associates

(Chiang and Melton, 2003). All these studies laid above demonstrate that

mature differentiated pancreatic cells derive from a common precursor that

expresses both IPF1/PDX1 and Ptf1α/p48.

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3.6.1 Specification of endocrine and exocrine cell fates by Notch

signalling

As mentioned above, early in pancreas development, the majority of

endocrine cells formed are glucagon-producing α-cells. From e12.5

onwards, the so-called “secondary transition”, endocrine cells differentiate

in exponentially increasing numbers, with β-cell differentiation

predominating (Pictet and Rutter, 1972; Herrera et al, 1991).

A key regulator of endocrine cell development is the basic-helix-loop-helix

(bHLH) transcription factor neurogenin 3 (ngn3). Ngn3 is expressed

exclusively by endocrine progenitors that descended from IPF1/PDX1-

expressing precursors and becomes down-regulated during differentiation

(Apelqvist et al, 1999; Grandwohl et al, 2000; Gu et al, 2004). Ngn3 is

normally expressed in a scattered fashion by cells within the epithelium and

ectopic expression of ngn3 in progenitor cells using the Ipf1/pdx1 promoter

results in total diversion of the pancreas to endocrine fate (Apelqvist et al,

1999; Schwitzgebel et al, 2000). Ectopic expression of ngn3 is also

sufficient to induce endocrine differentiation throughout the gut epithelium

(Grapin-Botton et al, 2001).

Ngn3 expression is reminiscent of other ngn-type bHLH transcription

factors broadly termed pro-neural (Lee 1997). In the developing nervous

system, the expression of these genes is controlled by a process called

lateral inhibition. Lateral inhibition is a classic way of specifying a particular

cell fate within a field of initially equivalent cells and is mediated by the

Notch signalling pathway (Lewis 1996; Beatus and Lendahl, 1998; Bray

1998; Figure 3). Notch signalling involves cells expressing high levels of the

ligands, Delta or Serrate signal to neighbouring cells that bear the Notch

receptor, thereby triggering the activation of the receptor and subsequent

cleavage of its intracellular domain. Intracellular Notch (NIC) prevents the

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signalling cell from adopting a primary fate, by interacting with the DNA-

binding protein RBP-Jκ and subsequent induction of transcription of bHLH

repressor genes, i.e. the Hes genes, which in turn repress expression of

downstream target genes, such as the ngn genes, that could promote the

primary cell fate (Lewis 1996; Beatus and Lendahl, 1998; Bray 1998).

This paradigm also applies to the pancreas (Figure 3). Cells that secrete

the ligands Delta or Serrate, hence Notch is not activated, can freely

express ngn3 and adopt the endocrine cell fate (primary cell fate). In

contrast, cells in which Notch is activated , Hes 1 expression is induced

leading to repression of ngn3 expression. These cells remain as

undifferentiated pancreatic progenitors (secondary fate).

Figure 3. Notch signaling in pancreatic cell type specification (see text).

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In effect, mice lacking Delta1, RBP-Jκ or the notch target gene Hes1

exhibit a phenotype where there is widespread premature endocrine

differentiation, with concomitant depletion of pancreatic progenitors

(Apelqvist et al, 1999; Jensen et al, 2000).

Murtaugh and associates have shown that timing of Notch signalling

activation is crucial for proper differentiation of pancreatic progenitors. By

establishing a modular transgenic model system, the authors concluded

that if Notch signalling becomes active in IPF1/PDX1-expressing precursor

cells both exocrine and endocrine differentiation is blocked and the cells

remain trapped in an undifferentiated state (Murtaugh et al, 2003). If Notch

signalling is activated after endocrine cell specification is completed, there

is no effect caused by this activation since fully differentiated cells become

non-responsive to Notch.

3.6.2 Factors involved in endocrine cell differentiation and

specification

NeuroD

NeuroD is a bHLH transcription factor that is expressed by several neuronal

cell types and plays a role in the induction of the neuronal cell

differentiation program. (Lee et al, 1995). In the pancreas, NeuroD is

expressed in all differentiated endocrine cells after they have undergone

differentiation and cell cycle arrest. NeuroD is, however, not critical for

endocrine differentiation since all endocrine cell types still form in NeuroD

mutant mice. However, islet cell number is gradually reduced in the mutant

mice and β-cells undergo apoptosis prior to birth, the mechanism of which

is currently unknown. Mutations in NeuroD are also associated with

maturity onset diabetes of the young 6 (MODY 6) in humans. The

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downstream functions of NeuroD are not well understood other than its

implication in insulin gene transcription. NeuroD is autoregulated

suggesting a role in the stabilization of the endocrine fate (Yoon et al,

1998). Although it is not required for endocrine differentiation, adenovirus-

mediated transfection of NeuroD in human ductal cells can initiate the

endocrine program (Heremans et al, 2002). The expression of NeuroD

precedes that of other endocrine post-mitotic markers such as Pax6 and

ISL1 suggesting a role for NeuroD in promoting cell cycle exit (Jensen et al,

2000).

ISL1

Immediately after establishment of the endocrine fate followed by cell cycle

arrest, the LIM-domain gene Isl1 becomes expressed. As mentioned

earlier on, Isl1 has an early function in mesenchyme formation and/or

survival. The Isl1 mutant mice exhibit complete dorsal pancreas agenesis

that most likely reflects the requirement for the surrounding mesenchyme in

pancreatic epithelial cell growth (Ahlgren et al, 1997). Since ISL1 is not

expressed in the early ventral mesenchyme, the ventral mesenchyme is

intact in the Isl1 mutant mice and ventral exocrine development is thus not

perturbed (Ahlgren et al, 1997). The addition of wild type mesenchyme from

pancreas or lung to explants of dorsal pancreatic epithelium derived from

the Isl1 mutant mice results in the development of exocrine tissue. Wild

type mesenchyme, could not, however, rescue endocrine cell differentiation

of Isl1-/- epithelia demonstrating that Isl1 function is required for endocrine

cell differentiation (Ahlgren et al, 1997). Later in development ISL1

expression is restricted to postmitotic endocrine cells excluding any

involvement of ISL1 in the selection of endocrine progenitor cells. However,

the precise mechanism of ISL1 function is still elusive but could be linked to

endocrine cell survival.

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Nkx genes

β-Cell competence cues, i.e the intrinsic or extrinsic molecules that promote

the β-cell fate, are likely to include the NK-homeodomain genes Nkx2.2 and

Nkx6.1 (Sussel et al, 1998; Sander et al, 2000). Nkx2.2 is expressed early

in the epithelium slightly after IPF1/PDX1 activation and its expression

persists during precursor cell expansion but it is later restricted to endocrine

α-, β- and PP cells. Targeted deletion of Nkx2.2 has no effect on the

phenotype or expansion of precursor cells but leads to impaired maturation

of endocrine cell types. Sussel and associates found that in the Nkx2.2

mice, although ngn3 expression appears normal, β-cells fail to activate the

insulin gene and display loss of Nkx6.1 suggesting that Nkx2.2 is essential

for specification of the mature β-cell phenotype (Sussel et al, 1998).

Nkx6.1, which is expressed both in pancreatic progenitor cells and later in

the differentiated β-cells of the mature pancreas (Jensen et al, 1996) is

another potential target for IPF1/PDX1. Nkx6.1 expression is initiated by

both Nkx2.2 and IPF1/PDX1 (Watanada et al, 2000) and conditional

mutation of Ipf1/pdx1 in the β-cells in mice leads to loss of Nkx6.1

expression (Ahlgren et al, 1998). Nkx6.1 is expressed in the dorsal and

ventral buds shortly after IPF1/PDX1 activation. It is also expressed in the

nervous system where it is involved in neuronal fate specification (Vallstedt

et al, 2001). In the endoderm, Nkx6.1 is restricted to cells committed to the

pancreatic fate. Nkx6.1 mutants show no obvious defects within the

pancreatic precursors suggesting that Nkx6.1, like Nkx2.2, is indispensable

for embryonic pancreatic growth (Sander et al, 2000). However, Nkx6.1 is

essential for β-cell formation as there is complete absence of mature β-cells

but normal development of α-cells (Sander et al, 2000). Like in Nkx2.2

mutants, ngn3 expression is normal in the Nkx6.1 mutants. Hence, Nkx6.1

is critical for the stabilization of the β-cell phenotype rather than induction of

the endocrine program.

25

Pax genes

Pax4 and Pax6 are both independent members of the paired-box

homeoprotein Pax gene family of transcription factors (Noll, 1993; Dahl et

al, 1997). Gene inactivation studies for Pax4 and Pax6 have demonstrated

the requirement for these two genes in pancreatic endocrine development

(St-Onge et al, 1997; Sosa-Pineda et al, 1997). Pax4 homozygous mutant

mice die three days after birth and immunohistochemical analysis of the

pancreas of newborn mice revealed complete absence of both β- and δ-

cells (Sosa-Pineda et al, 1997). α-Cells were present though but appeared

dispersed. These observations suggest that the main role of Pax4 is to

control β- and δ-cell development possibly by suppressing the molecular

cues that specify the α-cell fate.

Pax6 has been shown to be important for islet cell development. The

expression of Pax6 is restricted to the eye, the central nervous system, the

nose and the endocrine pancreas (Walther and Gruss, 1991; Turque et al,

1994). Pax6 is expressed early in the epithelium of the developing

pancreas at e9 in both dorsal and ventral buds and later on expression of

Pax6 is detected in all differentiated endocrine cell types. Mice with a

spontaneous Pax6 mutation, known as Small eye (Sey -/-), show abnormal

islet organization with decreased α-, β-, δ- and PP-cells (Sander et al,

1997). Pax6 binds and transactivates the promoters of the glucagon, insulin

and somatostatin genes suggesting a putative explanation to the decreased

hormone production observed in the Small eye mice (Sander et al, 1997).

Pax6 null mice show disorganized islets and die within minutes after birth

(St-Onge et al, 1997). A role of Pax6 in the regulation of cell adhesion has

been proposed with respect to the islet disorganization observed in the

Pax6 mutant mice (St-Onge et al, 1997; Chalepakis et al, 1994). Null

26

mutants of both Pax4 and Pax6 implicate these transcription factors as key

regulators of the terminal differentiation steps of the endocrine pancreas.

3.6.3 Factors involved in exocrine cell differentiation and

specification

Ptf α/p48 1

Studies by Petrucco and associates into genes expressed by exocrine cells

led to the identification of an exocrine-specific promoter binding complex

termed PTF1 (Petrucco et al, 1990). As already mentioned, the PTF1

complex consists of possibly two different bHLH A/B- class heterodimers

between the B-class bHLH protein Ptf1a/p48 and the ubiquitous A-member

E2A or HEB (Rose et al, 2001).

Ptf1a/p48 gene encodes for a tissue-specific protein (Ptf1/p48 protein)

shown to be essential for exocrine pancreatic development. Mice deficient

in Ptf1α/p48 failed to form exocrine pancreas and differentiated α-cells

migrate to the spleen (Krapp et al, 1998). However, as mentioned earlier

on, Ptf1α/p48 has been shown to be expressed by pancreatic progenitors

as early as e9.5 suggesting other roles for Ptf1α/p48 beside exocrine

differentiation. Kawaguchi and coworkers generated a knock-in of the Cre-

recombinase into the Ptf1α/p48 gene causing its disruption thus allowing

lineage tracing (Kawaguchi et al, 2002). Ptf1a/p48-/- cells followed a

duodenal fate choice contributing to all lineages in the gut (Kawaguchi et al,

2002) indicating that Ptf1a/p48 is required for maintaining cells in the

pancreatic fate.

27

Mist1

MIST1 is also a member of the B-class of bHLH factors and forms

heterodimers with E2A factors binding to typical E-boxes (Lemercier et al,

1997). MIST1 is expressed strongly in the pancreas but also in

nonpancreatic tissues like the salivary gland and stomach, in exocrine and

chief cells respectively (Pin et al, 2000). In the pancreas, MIST1 is confined

to exocrine cells and absent in ducts (Yoshida et al, 2001). At e12.5, MIST1

expression becomes heterogeneous in the peripheral epithelial regions

destined to become exocrine cells. This pattern suggests that MIST 1 has a

role in stabilizing the exocrine fate (Pin et al, 2001). MIST1 null mice

display presence of immature exocrine cells and lesions (Pin et al, 2001).

The cells inside these lesions coexpress ductal and exocrine genes

supporting the argument that MIST1 is required for the maintenance of

exocrine identity (Pin et al, 2001).

28

4. Aims of this thesis:

The aim of this thesis was to analyse the molecular cues that control

pancreatic epithelial cell proliferation and also to study the role of

FGF1 signalling in pancreas development and β-cell function. The

specific aims, therefore, were:

To investigate the role of FGF10 in pancreatic epithelial cell

proliferation (Paper I).

To examine the role of FGF2 signalling in β-cells (Paper II).

To elucidate the role for Wnt signalling in pancreas

development, specifically with respect to pancreatic epithelial

cell proliferation (Paper III).

29

5. RESULTS AND DISCUSSION:

5.1 PART 1- FGF SIGNALLING IN PANCREATIC GROWTH

AND HOMEOSTASIS (PAPERS I & II)

5.1.1 Background

Fibroblast Growth Factor (FGF) signalling

Fibroblast Growth Factors and their receptors

FGF signalling plays a pivotal role in mouse embryogenesis and has been

implicated in the development of many organs that are dependent upon

mesenchymal-epithelial interactions, including the pancreas. FGFs are

small polypeptide growth factors, all of which share in common certain

structural characteristics. By and large, FGF signalling appears to regulate

significant aspects of embryonic development, wound healing and

tumourigenesis.

To date, 23 distinct FGF members have been discovered. FGF ligands can

induce mitogenic, chemotactic and angiogenic activity in cells of

mesodermal and neuroectodermal origin (Basilica and Moscatelli, 1992).

Defining features of members of the FGF family is a strong affinity for

heparin and HLGAGs (Burgess and Maciag, 1989) and also the presence

of a central core of 140 amino acids that is highly homologous between

different members. This central core folds into twelve antiparallel β-strands

that form a cylindrical barrel surrounded by the more variable amino- and

carboxy-terminal stretches (Zang et al, 1991).

30

FGFs elicit their effects in target cells by signalling through cell-surface,

tyrosine kinase receptors. Isolation of an FGF receptor capable of binding

to FGF1 in chick provided valuable information on the structure of the FGF

receptor protein (Lee et al, 1989). This receptor was a transmembrane

protein that contains three extracellular Ig-like domains-IgI, IgII and IgIII-,

an acidic region between IgI and II, a transmembrane domain and an

intracellular tyrosine kinase domain. The FGF receptor belongs to the Ig

superfamily of receptors that also include the platelet-derived growth factor

(PDGF)-α receptor (PDGFαR), PDGFβR and interleukin-1 receptor (IL-1R),

(Johnson et al, 1990).

Different FGF ligands have diverse effects in diverse target cells. The

different responses of target cells to the various FGFs imply that they

express different forms of the receptor. At present, there are four FGF

receptors encoded by four different genes, each with splice isoforms. FGF

receptors transmit extracellular signals to various cytoplasmic signal

transduction pathways through tyrosine phosphorylation. Upon ligand

binding and dimerisation, the receptors phosphorylate specific tyrosine

residues on their own and each other’s cytoplasmic tails (Lemmon and

Schlessinger, 1994). Phosphorylated tyrosine residues recruit other

signalling molecules to the activated receptors and propagate the signal

through many different downstream transduction pathways such as the

mitogen-activated protein kinase (MAPK) MAPK pathway, the Protein

kinase C (PKC) pathway and others (Pawson, 1995 and Figure 4).

31

Figure 4. FGF intracellular singalling cascades.

FGF signalling in the developing pancreas

The pancreas is an organ whose growth, branching morphogenesis, and

differentiation are dependent on mesenchymal-epithelial interactions, thus

implying a potential role for FGFs and/or other growth factors (Edlund,

2002). FGF-signalling plays a key role in the development of the mouse

embryo and has been implicated in the development of many organs that

are dependent on epithelial-mesenchymal interactions (Kato S, 1999;

Szebenyi and Fallon, 1999).

32

Several independent studies have pointed to a role for FGF signalling

during pancreatic development and well as β-cell function. Mice that

express a dominant negative FGFR2b under the control of the

Metallothionein-promoter (Celli et al, 1998), mice that lack FGFR2b (Revest

et al, 2001) or Fgf10 (Ohuchi et al, 2000; Bhushan et al, 2001), a high

affinity ligand for FGFR2b, all show pancreatic hypoplasia. In addition, in

vitro cultures of rat pancreatic anlagen have suggested that signalling via

the FGFR2b stimulates exocrine differentiation and pancreatic endocrine

progenitor cell proliferation (Miralles et al, 1999; Elghazi et al, 2002).

FGF signalling in the adult pancreas

β-Cells are dedicated to the biosynthesis and accurate processing of

hormone precursors to produce mature insulin, which they store in large

dense core secretory vesicles (LDCV) called β-granules. The fine control of

glucose homeostasis is heavily dependent on the appropriate and timely

release of insulin granules from the β-cells. Accordingly, more than 99% of

endogenous insulin is secreted via a tightly regulated pathway (Rhodes and

Halban, 1987). Secretion of insulin is mainly under the control of blood

glucose (Matchinsky, 1996). Glucose-stimulated insulin secretion (GSIS) is

initiated by glucose uptake mediated by GLUT2 and its subsequent

phosphorylation by glucokinase (GCK). Glucose-6-phosphate is then

metabolized through the glycolytic pathway and activates mitochondrial

metabolism to generate coupling factors, the best described of these being

ATP. A rise in cytoplasmic ATP/ADP ratio leads to closure of KATP

channels, depolarization of the plasma membrane and opening of voltage-

sensitive Ca2+ channels. Then, extracellular Ca2+ enters the cell and

triggers exocytosis of the insulin granules (reviewed in Efrat, 2004 and

Figure 5).

33

Figure 5. Schematic representation of Glucose-Stimulated Insulin Secretion (GSIS; see text; modified from Efrat, 1997)

After its release from the β-cells, insulin acts by binding to its cell surface

receptor, thus activating the receptor’s intrinsic tyrosine kinase activity

resulting in receptor autophosphorylation and phosphorylation of several

substrates. Tyrosine phosphorylated residues on the receptor itself and on

subsequently bound receptor substrates provoke docking sites for

downstream signalling molecules, including adapters, protein

serine/threonine kinases and exchange factors. These molecules

orchestrate the numerous insulin-mediated physiological responses

(Lizcano and Alessi, 2002).

FGF signalling has been shown to be required for normal β-cell function

and maintenance of normoglycaemia in adult mice (Hart et al, 2000). Over-

expressing a dominant negative form of FGFR1c under the control of the

34

IPF1/PDX1 promoter leads to diabetes in mice. Perturbed FGFR1c

signaling impairs the expression of Glut-2 and prohormone convertase 1/3

(PC1/3), an enzyme involved in processing proinsulin into mature insulin in

β-cells, and hence mice with attenuated FGFR1c signaling, denoted FRID1

mice, show impaired glucose sensing and proinsulin processing.

Furthermore, FGFR1c signalling appears to be required for proper

postnatal expansion of β-cells (Hart et al, 2000).

5.1.2 More is less: FGF10 in pancreatic epithelial cell

proliferation

FGFR2 signalling in the pancreas

Several independent studies indicate that signalling via FGFR2b is crucial

for proper pancreatic development (Elghazi et al, 2002; Pulkkinen et al,

2003). Using an antibody that recognises all isoforms of FGFR2, to

determine the expression pattern of FGFR2 in the developing pancreas, we

could demonstrate that FGFR2 was expressed in non-differentiated

epithelial cells between e9 and e13 and not in differentiated cells at these

stages (Paper I, Fig.1). At later stages, from e13 onwards until adult stages,

FGFR2 expression was limited to insulin-producing cells (Paper I and

Figure 6).

35

Figure 6. FGFR2 is expressed in the developing pancreatic epithelium. A-F: Confocal analyses of the expression if FGFR2 at e9.5 (A), e11.5 (B), e13 (C), e15 (D), neonatal (E, neo) and adult (F) stages show that FGFR2 is expressed in pancreatic progenitor cells at early stages of development but then becomes gradually restricted to pancreatic β-cells (Paper 1, pp186).

The FGFR2b high-affinity ligand FGF10 has been reported to be highly

expressed in the mesenchyme that surrounds the pancreatic buds at e10

(Bhushan et al, 2001) and we also observed a low-level expression in the

pancreatic epithelium at later embryonic stages as well as in adult islets

(Hart et al, 2000)

Overexpression of FGF10 in the pancreas leads to increased

epithelial cell proliferation

36

To begin to define the mechanism by which FGF10/FGFR2b signalling

controls pancreatic progenitor cell proliferation and differentiation, we

generated transgenic mice expressing Fgf10 under the Ipf1/Pdx1 promoter.

The transgenic mice were born alive but appeared dehydrated, consistently

smaller in size and hyperglycaemic compared to their control littermates

resulting in death within the first week after birth. Gross morphological

examination of 15-day old embryos and neonates revealed a

macroscopically malformed pancreas that appeared greatly increased in

size as well as more solid and condensed, unlike the characteristic loose

and fluffy structure observed in pancreas of wild type littermates (Paper I,

Fig. 2A-D). These findings show that persistent expression of Fgf10 in the

developing pancreas results in pancreatic hyperplasia that occurs

progressively with age as further histological analyses confirmed (Paper I,

Fig 3)

Pancreatic hyperplasia is indicative of perturbed epithelial cell proliferation.

By double-immmunohistochemical analyses using antibodies against the

ductal specific marker cytokeratin-7 and the mitotic marker phospho-

Histone H3 on e17 pancreases (Paper I, Fig. 4A-F), we determined the

extent of epithelial cell proliferation in the Ipf1/Fgf10 versus control mice.

Cytokeratin-7 was significantly expressed throughout the epithelium in

contrast to the more restricted, patchy expression observed in the wild type

pancreas (Paper I, Fig. 4A,D) indicating that the transgenic pancreas

consisted mainly of ductal epithelial cells. The number of phospho-Histone

H3 positive cells/pancreatic area was increased by 50% in e17 Ipf1/Fgf10

mice as compared to stage-matched controls and total pancreatic area was

increased by 30% in the transgenic mice at this stage. Together these data

provide evidence that the pancreatic hyperplasia observed in the Ipf1/Fgf10

transgenic mice results from sustained proliferation of pancreatic ductal

epithelial cells.

37

Overexpression of FGF10 perturbs endocrine and exocrine cell

differentiation

A common theme in developmental biology is that proliferation and

differentiation are mutually exclusive, meaning that they cannot occur in the

same cell simultaneously. Therefore, what are the effects of enhanced

proliferation on cell differentiation in Ipf1/Fgf10 mice? To address this

question we examined in detail the differentiated state of the pancreas of

transgenic pups and specifically we looked into the expression of

transcription factors, hormones and enzymes. Immunohistochemical

analyses of neonatal pancreas using antibodies specific for insulin,

glucagon and somatostatin (data not shown) confirmed that pancreatic

endocrine cell differentiation was severely perturbed in the transgenic mice

(Paper I, Fig. 5 A-B). In normal mice endocrine cells cluster into distinct

islets (Paper I, Fig. 5A). The Ipf1/Fgf10 transgenic mice displayed

drastically fewer (less than 1% of that of wild type pancreas) hormone-

producing cells that failed to cluster (Paper I, Fig. 5B). Consequently, the

expression of the transcription factor ISL1 was mainly expressed in few,

non-clustered cells corresponding to the few hormone-producing cells that

formed in the transgenic pancreata, as opposed to the expression detected

in clustered pancreatic endocrine cells of control pancreata (Paper I, Fig.

5C-D). Together these data provide evidence that pancreatic endocrine cell

differentiation is perturbed in Ipf1/Fgf10 transgenic mice. Furthermore, the

expression of exocrine enzymes like Carboxypeptidase A (CPA; Paper I,

Fig. 5E-F) was drastically perturbed in Ipf1/Fgf10 mice compared to that in

control pancreata. The condensed organization of the pancreatic epithelia,

perturbed pancreatic acinar formation, uniform cytokeratin-7 expression

and drastically reduced exocrine expression in Ipf1/Fgf10 mice suggest that

exocrine cell differentiation is equally perturbed in Ipf1/Fgf10 mice.

Markedly so, endocrine and exocrine differentiation are not perturbed in the

38

Fgf10-/- mice suggesting that the signals inducing cell fate determination are

still active in these mice whereas a increased and prolonged expression of

FGF10, such as in the Ipf1/Fgf10 mice, disrupts those signals resulting in

endocrine and exocrine differentiation defects. So, if these cells are not

differentiated endocrine or exocrine cells, what are they?

Overexpression of FGF10 in the pancreas perturbs Notch-mediated

lateral inhibition

The increased pancreatic epithelial cell proliferation and impaired

pancreatic cell differentiation observed in Ipf1/Fgf10 mice raise the

possibility that the majority of these pancreatic epithelial cells may

represent pancreatic progenitor cells. Single-cell transcriptional profiling of

early pancreatic epithelial cells (Chiang and Melton, 2003) together with the

expression profile of different pancreatic transcription factors (Edlund,

2002) suggests that early pancreatic stem and/or progenitor cells co-

express the transcription factors IPF1/PDX1, Nkx6.1, Nkx2.2 and

Ptf1α/p48. Ipf1/Fgf10 neonates displayed a uniform low-level pancreatic

epithelial expression of IPF1/PDX1 with only occasional high-level

IPF1/PDX1-expressing cells, the latter most likely correspond to the

sporadic of Ins-producing cells that appear in the Ipf1/Fgf10 mice (Paper I,

Fig. 5H). In contrast, control mice displayed strong IPF1/PDX1 expression

in the β-cells with only a low, barely detectable expression in other

pancreatic cell types (Paper I, Fig. 5G). The Nkx-transcription factors

Nkx6.1 (Paper I, Fig. 5I-J) and Nkx2.2. (not shown) were also expressed at

low levels in the epithelium of the Ipf1/Fgf10 mice. The pancreatic

transcription factor Ptf1a/p48 (Krapp et al, 1998), which is expressed in

early pancreatic progenitor cells from ~e10 (Selander and Edlund, 2002;

Kawaguchi et al, 2002) was also expressed throughout the pancreatic

39

epithelium of transgenic neonates (Paper I, Fig. 5 K-L). The expression of

IPF1/PDX1, Nkx6.1, Nkx2.2, and Ptf1α/48 in Ipf1/Fgf10 pancreatic

epithelial cells is supportive of an immature, progenitor-like nature of these

cells.

The presence of proliferating progenitor cells that failed to differentiate into

endocrine or exocrine cell types in the pancreas of the transgenic mice

implies that either the induction of pancreatic cell types or their terminal

differentiation is perturbed. To resolve this, we analysed the expression of

Notch signalling components in the transgenic pancreata by in situ

hybridisation. During pancreatic development ngn3 expression marks

endocrine progenitors and its expression is regulated via the Notch-

signalling pathway (Apelqvist et al, 1999; Jensen et al, 2000). Analyses

revealed that ngn3 expression was drastically reduced in e15 Ipf1/Fgf10

mice (Paper I, Fig. 6A-B) when compared to stage-matched littermates,

suggesting that pancreatic endocrine cell differentiation was impaired

already at the endocrine progenitor stage. Consistent with the reduced

number of ngn3 expressing cells, Dll1 expression also appeared reduced

as compared to wild-type pancreas (Paper I, Fig. 6C-D). How can we

explain this?

Apoptosis of early endocrine progenitors could be one explanation.

However, TdT-mediated dUTP nick-end labeling (TUNEL) assay and

immuno staining using that apoptotic marker Caspase 3 (data not shown)

did not detect any increase in cell apoptosis suggesting that the decreased

number of pro-endocrine and endocrine cells in Ipf1/Fgf10 mice is caused

by impaired specification of pro-endocrine cells rather than cell death.

Impaired specification of endocrine progenitors automatically refers to

negative regulators of ngn3 expression such as Notch1 and its downstream

target gene Hes-1. In the wild-type pancreas Notch1 was highly expressed

40

predominantly in the developing acini at e15 (Paper I, Fig. 6E-F) but absent

from the streaks of differentiating endocrine cells (data not shown and

Apelqvist et al, 1999). In the Ipf1/Fgf10 mice Notch1 expression appeared

uniform throughout the pancreatic epithelium, reminiscent of the expression

of Notch1 in e13 pancreatic epithelium (Apelqvist et al, 1999). The uniform

expression of Notch1 in the Ipf1/Fgf10 pancreatic epithelia that also

matched Hes1 expression (Paper I, Fig. 6G-H), suggests that Notch

activation is maintained in the pancreatic epithelium of Ipf1/Fgf10 mice.

Consequently, a uniform, maintained activation of Notch is likely to impair

pancreatic expression of ngn3 in Ipf1/Fgf10 mice. Together these data

show that persistent expression of Fgf10 affects Notch signalling. Hence,

the lack of differentiated cell types in the Ipf1/Fgf10 mice might reflect a

perturbed Notch-mediated lateral inhibition pathway where sustained Notch

activation would “lock” the majority of the pancreatic epithelial cells in a

non-differentiated, progenitor-like proliferating state.

However, the reduced expression of Dll-1 would imply that Notch is

activated via another mechanism different from lateral inhibition. This

distinct mechanism of Notch/Hes-1 activation seems to be linked to

precursor cell maintenance and not to pancreatic cell specification and

involves alternative ligands other than Dll-1. Candidate ligands are Jagged

1 and 2 (or Serrate 1 and 2). Both exhibit a uniform expression pattern in

epithelial cells of wild type e13 pancreas that overlaps with that of Notch 1

and Notch2 (Apelqvist et al, 1999; Lammert et al, 2000; Jensen et al,

2000). In a parallel study by Norgaard et al (2003) where FGF10 also was

overexpressed under the Ipf1/Pdx1 promoter, the authors showed that the

uniform expression of Jagged 1 and 2 is maintained in the transgenic mice

suggesting that Jagged 1 and 2 may activate Notch, thereby leading to

maintained proliferation of pancreatic progenitor cells.

41

Crosstalk between FGF and Notch pathways to regulate proliferation

versus differentiation occurs frequently during development in mice rather

than being a unique feature of the developing pancreas. During distal

outgrowth of the developing limb, FGF8 and 10 regulate expression of

Jagged 1 and 2 (Shawber et al, 1996; Valscecchi et al, 1997). Likewise,

FGF10 can maintain the dental epithelial precursor cell pool by stimulating

Hes-1 expression in the developing tooth (Mustonen et al, 2002; Harada et

al, 2002). Studies of neuronal differentiation in vitro suggest a similar

mechanism where FGFs stimulate proliferation and inhibit differentiation by

means of the Notch pathway (Faux, et al, 2001). Our data, therefore,

support a dual role of FGF10 in pancreas development: to maintain the

pancreatic progenitor cells in an undifferentiated state, involving directly or

indirectly the Notch signalling pathway, and to provide the proliferative cue.

Sel1-L role in the developing pancreas

The Sel-1l gene (Biunno et al, 1997; Biunno et al, 2000) is the mouse

homologue of the C.elegans sel-1 (suppressor-enhancer-lin) gene, which

encodes for a protein that acts as a negative regulator of lin12 (Grant and

Greenwald, 1997) the C.elagans homologue of Notch. Notch signalling is

critical for pancreatic endocrine and exocrine cell fate specification

(Apelqvist et al, 1999; Jensen et al, 2000) and it has also been implicated in

cell proliferation and apoptosis, and two vertebrate lin12/Notch

homologues, murine int-3 and human Tan-1 have been associated with

several cancers (Ellisen et al, 1991; Jhappan et al, 1992; Robbins et al,

1992; Capobianco et al, 1997; Shelly et al, 1999).

Sel-1l is abundantly in the pancreatic epithelium and more specifically in

the acini from e13 onwards (Biunno et al, 1997; Donoviel et al, 1998;

Harada et al, 1999; Cattaneo et al, 2001; our own unpublished

observations). A role for Sel-1l has been proposed in oncogenesis. Sel-1l

42

has been recently demonstrated to reduce both proliferative activity in vitro

and aggressiveness in patients with breast cancer (Orlandi et al, 2002) and

the ectopic expression of the entire Sel-1l cDNA significantly reduces the

proliferate activity and aggressive behaviour of the human breast

carcinoma cell line MCF-7 (Cattaneo et al, 2001). Moreover, Sel-1l has

been also found to be downregulated or completely repressed in a

proportion of primary adenocarcinomas of the pancreas (Biunno et al,

1997; Donoviel et al, 1998) suggesting that Sel-1l might play a role in

pancreatic cancer. However, the mechanism responsible for this biological

effect remains unclear but given the role of Notch in tumourigenesis, the

effect of Sel-1l on proliferative activity might be mediated by negative

regulation of Notch signalling.

Very little is known about the function of mouse or human Sel-1l. It has

been established that it implicating a role for Sel-1l during pancreatic cell

differentiation. Evidence from yeast and C.elegans indicate that the function

of Sel-1l is associated with the degradation or trafficking of several proteins

(Hampton et al, 1996). In C.elegans, Sel-1l is involved in signalling

pathways that maintain equilibrium between the capacity of ER to process

client proteins and the physiological demand of the organelle (Urano et al,

2002; Kaufman et al, 1999; Patil and Walter, 2001). Accumulation of

improperly folded proteins in the ER results in the activation of ER stress.

Pancreatic β-cells have a highly developed and active ER, because of

various and large amounts of ER client proteins (Harding et al, 2001).

Therefore, the health of these cells is closely linked to ER homeostasis.

Disruption of Sel-1l expression using RNAi technology resulted in perturbed

growth and metabolic activity of βTC-3 cells, a pancreatic β-cell line.

In contrast to the high level of pancreatic expression of Sel-1l in the

developing acini of the e15 control mice, Sel-1l was expressed at an

apparently lower level in the pancreas of stage-matched Ipf1/Fgf10

43

transgenic mice (Paper I, Fig. 6J). Together these findings suggest that

persistent ductal epithelial expression of Fgf10 impairs the expression of

the Notch antagonist Sel-1L. Hence, based on our and others data, we

propose a model (Figure 6) whereby FGF10, is predominantly expressed

in the mesenchyme, signals to the epithelium to stimulate proliferation of

early pancreatic progenitor cells. At later stages of development, as the

mesenchyme/epithelium ratio decreases, the FGF10 concentration may no

longer be sufficient to promote proliferation and consequently, possibly

involving increased expression of Sel-1l, progenitors escape the non-

differentiated state and enter the differentiation program.

Figure 7. Model of a possible crosstalk between FGF10 and Notch to regulate proliferation and differentiation of early pancreatic progenitors (see text).

It should also be stressed that although Fgf10 appears to be crucial for

pancreatic growth (Ohuchi et al, 2000; Bhushan et al, 2001) other factors,

including other FGFs and the EGF-family of signalling factors, have also

been implied in pancreatic cell proliferation (reviewed in Edlund, 2002),

indicating that FGF10 may act in concert with other factors to effectively

stimulate pancreatic progenitor cell proliferation.

44

5.1.3 Enough is Enough: Persistent FGF signalling in β-cells

leads to diabetes in mice

Overstimulation of FGF activity in mice leads to diabetes

Apart from playing a key role in pancreatic epithelial cell proliferation, FGF

signalling, via FGFR1c in particular, has been previously shown to be

critical also for adult β-cell function (Hart et al, 2000). Overexpression of a

dominant negative form of FGFR1c under the Ipf1/Pdx1 promoter leads to

diabetes in mice due to severely perturbed glucose uptake and insulin

processing. Moreover, the postnatal expansion of β-cells was reduced in

mice with perturbed FGFR1c signalling (Hart et al, 2000). To further dissect

the mechanism by which FGFR1c controls β-cell function and β-cell number

we over-expressed the FGFR1c high-affinity ligand FGF2 in pancreatic β-

cells in mice. As mentioned in the Introduction, FGF2 is expressed early in

development in the notochord and has been reported to be implicated in

the induction of the dorsal pancreatic program (Hebrok et al, 1998) by

suppressing shh expression in the presumptive pancreatic endoderm. In

addition, FGF2 is expressed in adult β-cells (Hart et al, 2000). Ipf1/Fgf2

neonates were born alive and presented with a well-developed pancreas

indistinguishable from that of their littermates. However, at approximately

10-12 weeks of age, elevated blood (Paper II, Fig. 1A) and urine glucose

(data not shown) levels were observed indicative of diabetes, suggesting

that persistent FGFR1c signalling in the adult pancreas leads to impaired

glucose homeostasis.

The diabetic phenotype of the Ipf1/Fgf2 mice was further examined by

monitoring serum insulin levels at fasted conditions in response to glucose

challenge (Paper II, Fig. 1B). Ipf1/Fgf2 mice showed significantly lower

serum insulin levels and both first and second phase insulin release were

45

perturbed in these mice compared to control littermates (Paper I, Fig. 1B).

Together these results demonstrate that Ipf1/Fgf2 mice fail to secrete

enough insulin in response to elevated blood glucose levels providing

evidence that persistent FGF activation in β-cells severely perturbs GSIS.

Perturbed GSIS could mean: a) decreased amounts of stored insulin or b)

insulin resistance c) perturbed glucose uptake and metabolism and d)

combination of some or all of the above. To determine which one, we first

measured the total insulin content/pancreatic protein, i.e relative total

insulin content and observed a ~30% reduction, albeit not statistically

significant, in the pancreas of Ipf1/Fgf2 mice compared to control

littermates (Paper II, Fig. 2A). The transgenic mice responded normally to

exogenous insulin administration (Paper II, Fig. 2B) suggesting that neither

decreased amounts of stored insulin nor insulin resistance were the primary

causes of the perturbed GSIS displayed by the Ipf1/Fgf2 mice.

Persistent FGF signalling disrupts islet morphology and perturbs

expression of key regulatory genes of β-cell function and

homeostasis.

In our quest for the cause(s) for the diabetes in the Ipf1/Fgf2 mice, we

looked into the expression of genes critical for adult β-cell function using

immunohistochemistry and real-time RT-PCR. The transcription factors

IPF1/PDX1, ISL-1 and Nkx6.1, the hormones insulin and glucagon and the

exocrine enzyme carboxypeptidase A (CPA) were all expressed in the

pancreas of the transgenic mice (Paper II, Fig .2A-B, I-J, K-L, M-N, O-P).

The islets of the Ipf1/Fgf2 mice showed, however, an atypical organisation

where α-cells appear scattered throughout the islets as opposed to their

normal peripheral location in age-matched wt littermates (Paper II, Fig. 2A-

46

B). Together these data indicate that overexpression of FGF2 perturbs islet

morphology but does not affect endocrine of exocrine cell differentiation.

Perturbed insulin secretion could result from defect glucose uptake by β-

cells mediated by Glut2. Glut2 is expressed at low levels in fetal β-cells and

becomes progressively increased after birth. To assess whether perturbed

Glut2 expression contributed to the blunted GSIS in the Ipf1/Fgf2 mice, we

compared Glut2 expression in β-cells of Ipf1/Fgf2 and wt mice. Both

immunohistochemical (Paper II, Fig. 3K-L) and real-time RT-PCR (Paper II,

Fig. 4) analyses revealed dramatically reduced Glut2 expression levels

consistent with the hyperglycaemia, glucose intolerance and perturbed

insulin secretion observed in Glut2 -/- mice (Guillam et al. 1997) when

challenged with exogenous glucose. Our data, therefore suggest that

closely monitored FGF signalling is required for high-level Glut2 expression

in postnatal β-cells. But what might be the link between FGF signalling and

Glut-2 levels in adult β-cells?

Work by us (Ahlgren et al, 1998; Hart et al, 2000) and others (Wang et al,

2000; Boj et al, 2001; Hagenfeldt-Johansson et al, 2001; Shih et al, 2001;

Yamagata et al, 2002) have proposed transcription factors IPF1/PDX1 and

HNF1α to be two key regulators of Glut2 expression. Hence, to investigate

whether the impaired Glut2 expression observed in the Ipf1/Fgf2 mice was

the consequence of reduced expression of Ipf1/Pdx1 and/or HNF1α, we

monitored their expression by real-time RT-PCR. Ipf1/Pdx1 expression was

not affected in the transgenic mice whereas HNF1α expression appeared

reduced by almost 80% compared to wild-type littermates (Paper II, Fig. 4).

The drastically blunted GSIS observed in the Ipf1/Fgf2 mice (Paper II, Fig.

1B) indicated that the insulin secretory mechanism of the Ipf1/Fgf2 mice

might be perturbed also downstream of Glut2 expression and glucose

47

uptake. The catalytic action of GCK during the conversion of glucose to

glucose-6-phosphate is rate-limiting for glucose metabolism because it

adjusts the amounts of secreted insulin to extracellular glucose levels

(Matschinsky, 2002). GCK expression examined by both

immunohistochemistry (Paper II, Fig. 3M-N) and real time RT-PCR (Paper

II, Fig. 4) appeared drastically reduced in transgenic mice compared to that

of the controls indicating that glucose sensing and metabolism are severely

perturbed in the Ipf1/Fgf2 mice.

Uncoupling protein-2 (Ucp-2) impairs GSIS by uncoupling respiration from

oxidative phosphorylation, which in turn leads to reduced ATP-production

(Chan et al, 1999; Zhang et al, 2001). Over-expression of Ucp-2 in β-cells

disrupts GSIS (Chan et al, 1999; Chan et al, 2001), whereas both Ucp-2 +/-

and -/- mice show improved GSIS (Zhang et al, 2001; Saleh et al, 2002).

The mechanism by which Ucp-2 expression and/or activity is regulated

remains however largely unknown although recent data suggest that

superoxide can stimulate UCP-2 activity (Krauss et al, 2003). Ucp-2

expression was increased by ~400% in the mRNA of islets derived from the

Ipf1/Fgf2 mice as compared to control islets (Paper II, Fig. 4), which is likely

to impair the generation of ATP in β-cells of Ipf1/Fgf2 mice. Collectively

these data provide evidence that enhanced FGF2 activity leads to a

decreased ATP/ADP ratio as a consequence of the reduced expression of

Glut2 and GCK as well as the concomitant increased expression of Ucp-2

in β-cells. Thus, persistent FGF signalling leads to perturbed expression of

key genes involved in glucose uptake, glucose metabolism and ATP

generation in β-cells.

FGF signalling negative feedback mechanisms in the β-cells?

48

The phenotype of the Ipf1/Fgf2 mice is reminiscent of that of the FRID1

mice. Both display diabetes and near complete loss of Glut2. However,

PC1/3 expression is unaffected in the Ipf1/FGF2 mice (Fig) but appears

severely downregulated in the FRID1 mice (Hart et al, 2000). HNF1a

expression is not changed in the FRID1 mice (own unpublished data)

whereas it is severely reduced in the Ipf1/Fgf2 mice suggesting that Glut-2

downregulation in these two mouse models is achieved either by two

different distinct mechanisms or by affecting different components of the

same mechanism. Having said that, the differences in the phenotypes

between these mouse lines could be attributed to transgene expression

levels or to the fact that, apart from FGFR1c, FGF2 can act via other

FGFRs (Ornitz and Itoh, 2001), for example FGFR2c, which was previously

reported to be expressed in the pancreas (Dichmann et al, 2003). On the

other hand, the similarities in the phenotype between the FRID1 and

Ipf1/Fgf2 mice might imply the presence of feedback mechanisms in the β-

cells. What could these mechanisms be and how would they enable β-cells

to ensure appropriate levels of FGF signalling?

FGF signalling induces a number of complex intracellular systems (Niehrs

and Meinhardt, 2002; Hacohen et al, 1998; Furthauer et al, 2002; Tsang et

al, 2002; Kovalenko et al, 2003; Wakioka et al, 2001; Lax et al, 2002) that

negatively regulate the pathway. Example of such systems are the Sprouty

proteins, Pyst1/MKP3, MKP1 and Sef which bind to different downstream

components of the FGF signalling cascade and block transmission of the

signal to the nucleus (Tsang and Dawid, 2004; Figure 8).

49

Figure 8. Feedback inhibitors of FGF signalling. Sef interacts with FGFRs and prevents phosphorylation of FRS2. A second less-defined mechanism suggests that Sef blocks FGF signalling downstream of MEK, and this is also the case for the intracellular splice variant Sef-b. Spry inhibits FGF signalling by sequestering Grb2, preventing its binding to FRS2; another inhibitory mechanism involves direct interaction of Spry with Raf. MKPs are negative regulators that work by desphosphorylating activated ERKs. MKP3 functions within the cytoplasm, whereas MKP1 is localised in the nucleus (modified from Tsang and Dawin, 2004).

The first bona fide feedback regulator of the FGF pathway, Sprouty (Spry),

was discovered through a genetic screen in Drosophila (Hacohen et al,

1998). FGF signalling controls airway branch formation and spry mutants

display ectopic airway branches due to excessive FGF signalling. Spry

proteins are widely conserved, with four members identified in mammalian

species (Cabrita and Christofori, 2003). Overexpression and gene depletion

studies in zebrafish and mouse have confirmed that vertebrate Sprys are

functionally conserved and antagonize FGF signalling (Minowada et al,

1999; Mailleux et al, 2001; Furthauer et al, 2001). We examined the

expression of all four mouse spry genes in the islets of Ipf1/Fgf2 and wt

50

mice using real time RT-PCR (Paper II, Fig. 4). Spry-1 expression was not

affected in the transgenic mice whereas expression levels of Spry-2

appeared increased almost 5-fold in the Ipf1/Fgf2 islets compared to the

controls (Paper II, Fig. 4). No expression of either Spry-3 or Spry-4 was

detected (data not shown). These data suggest that FGF2 overexpression

leads to Spry-2 upregulation, an inhibitor of FGF signalling.

FGF signalling is mediated via tyrosine kinase receptors that can act

through a number of transduction pathways including the highly conserved

Ras-ERK-MAPK signalling cascade (Martin 1998; Umbhauer et al, 1995;

Michelson et al, 1998; Borland et al, 2001). Biochemical assays and studies

in cultured mammalian cells have identified phosphatases that specifically

act on the ERK1/2 MAP kinases (Keyse, 2000). One of those enzymes,

Pyst1/MKP3, binds selectively to ERK2, which results in catalytic activation

of the phosphatase. Expression of Pyst1/MKP3 in mammalian cells

specifically blocks activation and nuclear translocation of ERK2 (Groom et

al, 1996; Camps et al, 1998; Brunet et al, 1999). These observation

strongly suggest that Pyst1/MKP3 acts as an intracellular brake to signal

transduction via the Ras/MAPK pathway in mammalian cells. In chick

embryos, overexpression of Pyst1/MKP3 causes limb bud truncations and

defects in neural plate development suggesting that FGF-inducible

expression of Pyst1/MKP3 is critical in maintaining appropriate levels of

signalling through the Ras/MAPK cascade (Eblaghie et al, 2003).

Moreover, Pyst1/MKP3 is co-expressed with FGF signalling in many known

sites throughout the mouse embryo suggesting that Pyst1/MKP3 regulates

FGF signalling during vertebrate development (Dickinson et al, 2002).

Therefore, we examined the expression of Pyst1/MKP3 in islets of

transgenic and wild type mice by real time RT-PCR and observed a 2-fold

increase in its expression in the Ipf1/Fgf2 mice compared to wild-type

littermates (Paper II, Fig. 4). Altogether these data suggest a model

whereby overstimulation of FGF signalling caused by persistent FGF2

51

expression leads to upregulation of Spry-2 and Pyst1/MKP3, a subsequent

block of FGF signalling and ultimately diabetes. In addition, given the way

these FGF inhibitors work, it seems plausible that FGF2 transduces its

effects via the Ras/MAPK pathway (Figure 9).

Figure 9. IPF1/PDX1 induces the expression of FGFR1c (1). Excess FGF2 binds to FGFR1c and stimulates downstream transduction cascades -possibly the MAPK pathway- (2-3). FGFR1c signalling induces transcription of genes that encode for the FGF inhibitors Spry-2 and Pyst1/MKP3 (4). Spry-2 and Pyst1/MKP3 the block FGFR1c signalling downstream of the receptor (5) resulting in perturbed glucose sensing (by Glut-2 and GCK downregulation), glucose metabolism (by UCP2 upregulation) and ultimately impaired insulin secretion (modified from Edlund, 2001).

52

5.2 PART 2- WNT SIGNALLING IN PANCREATIC GROWTH

AND HOMEOSTASIS (PAPER III)

5.2.1 Background

Wnt signalling Wnt ligands and Frizzled receptors- the basics

Wnt are secreted glycoproteins implicated in a variety of developmental

processes such as cell differentiation, cell polarity, cell migration and cell

proliferation among others (Peifer and Polakis, 2000; Novak and Dedhar,

1999), and are well studied in the fly, worm, fish, frog, mouse and human

(Wodarz and Nusse, 1998). At least 18 different Wnts have been identified

in the mouse alone, a diversity resulting from alternative splicing and

alternative promoter activation.

Wnts are thought to activate the function of members of the Frizzled (Frz)

gene family (Bhanot et al, 1996; Yang-Snyder et al, 1996). Frz are

serpentine receptor proteins and consist of seven transmembrane-

spanning domains, an N-terminal region and a cytoplasmic C-terminal

domain with sites suitable for phosphorylation (Bhanot et al, 1996; He et al,

1997; Wang et al, 1996). Frz proteins show an overall homology to

members of the superfamily of seven-transmembrane receptors known to

signal via heterotrimeric G-proteins, i.e G-protein-coupled receptors (Dale,

1998). Downstream target of Frz actions can be segregated into two types

of pathways: the canonical, which acts through the protein β-catenin to

regulate transcription; the non-canonical, a term referring to several

different cascade systems independent of β-catenin such as the planar cell

53

polarity (PCP) pathway that regulates Drosophila development and the

Wnt/Ca2+ pathway (Figure 10).

Figure 10. A schematic representation of the Wnt signal transduction cascade. (a) For the canonical pathway, signaling through the Frizzled (Fz) and LRP5/6 receptor complex induces the stabilization of β-catenin via the DIX and PDZ domains of Dishevelled (Dsh) and a number of factors including Axin, glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). β-catenin translocates into the nucleus where it complexes with members of the LEF/TCF family of transcription factors to mediate transcriptional induction of target genes. β-catenin is then exported from the nucleus and degraded via the proteosomal machinery. (b) For non-canonical or planar cell polarity (PCP) signaling, Wnt signaling is transduced through Frizzled independent of LPR5/6. Utilizing the PDZ and DEP domains of Dsh, this pathway mediates cytoskeletal changes through activation of the small GTPases Rho and Rac. (c) For the Wnt-Ca2+ pathway, Wnt signaling via Frizzled mediates activation of heterotrimeric G-proteins, which engage Dsh, phospholipase C (PLC; not shown), calcium-calmodulin kinase 2 (CamK2) and protein kinase C (PKC). This pathway also uses the PDZ and DEP domains of Dsh to modulate cell adhesion and motility. Note that for the PCP and Ca2+ pathways Dsh is proposed to function at the membrane, whereas for canonical signaling Dsh has been proposed to function in the cytoplasm (modified from Habas and Dawid, 2005)

54

Among the members of the Wnt family, Drosophila wingless is arguably the

best characterized. Based on the wingless mutant phenotype, genetic

screens performed in Drosophila have identified many components of

Wingless signalling and epistasis analyses have established a universal

pathway of Wingless signal transduction (Cardigan and Nusse, 1997;

Wodarz and Nusse, 1998). In addition, studies of Wnt signalling in

C.elegans, Xenopus and mouse have largely confirmed the finding in

Drosophila and expanded our understanding of the canonical Wnt

signalling pathway. In a simplified model for the canonical Wnt pathway,

Wnt ligands are secreted from expressing cells to reach their target cells. At

the target cells, Wnts bind to frizzled receptors (Bhanot et al, 1996; Yang-

Snyder et al, 1996; He et al, 1997) and co-receptor low-density-lipoprotein

(LDL)-receptors 5 and 6 (LRP5/6; Pinson et al, 2000; Wehrli et al, 2000;

Tamai et al, 2000; Schweizer and Varmus, 2003). Through a, by far,

unknown mechanism, the binding of Wnts with their receptors induces the

phosphorylation of the cytoplasmic protein Dishevelled (Yanagawa et al,

1995) which acts by relieving the constitutive repression by Wnt signaling

inhibitors. Downstream of Dishevelled, Axin is one of those inhibitory

proteins. In the absence of Wnt, Axin acts as a scaffold that brings together

glycogen synthase kinase 3 (GSK3) and β-catenin. This facilitates the

phosphorylation of β-catenin which is subsequently targeted for

ubiquitination and proteosome-mediated degradation (Arbele et al, 1997).

On the other hand, in the presence of Wnt, Dishevelled prevents the

phosphorylation of β-catenin, thereby stabilizing it (Yanagawa et al, 1995;

Riggleman et al, 1990; Hinck et al, 1994). Consequently, Armadillo/β-

catenin accumulates in the cytoplasm, translocates into the nucleus and

interacts with members of the T-cell /lymphoid enhancer factor (TCF/LEF)

family of architectural HMG box transcription factors (Molennar et al, 1996;

Behrens et al, 1996). The complex binds to consensus TCF/LEF sites in

55

promoters and induces transcription of Wnt-responsive genes (Behrens et

al, 1996; Riese et al, 1997; Brannon et al, 1997).

The most robust and compelling evidence for the presence of a β-catenin-

independent or non-canonical Wnt pathway came from studies of

gastrulation movements in vertebrates. Furthermore, there are indications

that vertebrate non-canonical Wnt signalling may also regulate processes

as diverse as cochlear hair cell morphology, heart induction, dorsoventral

patterning, tissue separation, neuronal migration and even cancer (Veeman

et al, 2003). Potential mechanisms of non-canonical Wnt signalling

transduction are also quite diverse, including signaling through intracellular

Ca2+ flux, janus kinase (JNK) and both small and heterotrimeric G proteins.

Vertebrate non-canonical Wnt signaling requires Frz receptors and the

proteoglycan co-receptor Knypek. Like the canonical Wnt signalling, this

pathway involves Dsh, although in this case, Dsh is localized to the cell

membrane via its DEP domain.

Wnt signalling in the pancreas

Little is known about the role of Wnt signalling during pancreas

development but current evidence suggests that Wnts may play a

fundamental role in both early and late stages of development as well as in

adult islets. Frz receptors have been found to be abundantly expressed in

the pancreas. Heller and associates (2002) have shown by RT-PCR that

Wnt 5a, 5b, 2b, 7b, 11 as well as Frz 2-9 transcripts are expressed in the

mouse pancreas, with expression levels peaking before e15 in the mouse,

i.e before endocrine differentiation reaches its climax and the mesenchyme

is markedly reduced. By in situ hybridization, Wnt expression was found

mainly in the mesenchyme but also in the islets at adult stages, with the

receptors Frz expressed in the endocrine cells later in development. Wnts

are expressed in the mesenchyme and signal to the epithelium in several

56

organ systems (Nusse and Varmus, 1992). The presence of Wnts in the

pancreatic mesenchyme at early stages once again supports a role for

mesenchymal-epithelial interactions in inducing branching morphogenesis

and in facilitating indirectly endocrine cell differentiation. Moreover, Wnt

expression in the pancreas seems to be conserved between mice and

humans. Heller and associates analysed human exocrine pancreas as well

as isolated islets by RT-PCR and reported the expression of a large

number of Wnt and frizzled proteins (Heller et al, 2002).

Functional data have also emerged on a role of Wnt signalling in

endodermal patterning and differentiation in mice. Transgenic mice where

Wnt-1 and Wnt5a are mis-expressed under the Ipf1/Pdx1 promoter show

perturbed pancreatic development (Heller et al, 2002). In Ipf1/pdx1-wnt1

embryos, the stomach to duodenum transition was shifted and pancreas

and spleen development were arrested or absent, while in Ipf1/pdx1-wnt5a

embryos, the pancreas appeared greatly reduced in size with impaired

architectural and histological appearance (Heller et al, 2002). The

differences in the phenotype observed in these two transgenic models

could be attributed to differences in binding of Wnt1 and Wnt5a to subtypes

of Frz receptors thus activating different downstream cascades. In addition,

further evidence has suggested that Wnt signalling may have a significant

role in the normal function of adult β-cells. Transcripts of Wnt2b and Wnt4

genes have been reported in adult islets by RT-PCR and gene profiling

approaches respectively (Heller et al, 2002; Gu et al, 2004). Furthermore,

LRP5, a coreceptor for canonical Wnts, was shown to be essential for

glucose-induced insulin secretion (Fujino et al, 2003). LRPs are

transmembrane proteins implicated in Wnt signalling in Xenopus (Wehrli et

al, 2000), Drosophila (Tamai et al, 2000) and mice (Pinson et al, 2000).

Studies in these organisms have suggested that LRP5 and LRP6 function

as co-receptors for Wnt ligands. Islets of mice deficient in LRP5, showed

glucose intolerance and impaired glucose-stimulated insulin secretion due

57

to reduced levels of ATP and Ca2+ (Fujino et al, 2003). Additionally,

exposure of wild-type islets to soluble Wnt3a and Wnt5a induced glucose-

stimulated insulin-secretion and the secretion was blocked by addition of

soluble Wnt inhibitor protein Frizzled-related protein 1 (FRP-1).

Finally, Wnts, when aberrantly activated, can lead to pancreatic tumour

formation. Molecular studies have pinpointed activating mutations of the

canonical Wnt signalling pathway as the cause of approximately 90% of

colorectal cancer. Ironically enough, Wnt ligands themselves are only rarely

involved in the activation of the pathway during carcinogenesis. Mutations

mimicking Wnt stimulation- by activation of APC (APC is found in a

complex with Axin and GSK3β that sequesters β-catenin) and β-catenin

mutations- result in nuclear accumulation of β-catenin, which subsequently

interacts with TCF/LEF factors to activate gene transcription (Giles et al,

2003). Indeed, mutations in APC have been implicated in the development

of pancreatic cancers in humans (Miao et al, 2003; Koesters and von

Knebel Doeberitz, 2003; Tanaka et al, 2001) and mice (Cullingworth et al,

2002). These findings reveal a role for canonical Wnt pathway in

proliferation of pancreatic epithelial cells and that tight regulation of this

pathway is essential for maintenance of the differentiated state.

5.2.2 Less can be lesser: Wnt signalling in pancreatic growth and

function

Wnts and Frzs are expressed in the pancreas

We used degenerate RT-PCR approach to screen for Wnts and Frz in the

mouse embryonic pancreas of stage e12 to e15 (data not shown). Wnt4,

wnt7b and frz3 were among those isolated and DIG-labelled probes

corresponding to those genes were designed to examine their spatial and

58

temporal expression during pancreas development by in situ hybridization

(Figure 11).

Figure 11. Wnt4 and Frz3 expression in the developing and neonatal pancreas (A-F). (A-C) In situ hybridisation of e10, e13, and neonatal pancreas and e13 stomach (insert in B) using a DIG-labelled Wnt4 probe (dark blue in A and C; green pseudo-colour in B), counterstained with antibodies against ngn3 (red) in B). (D-F) In situ hybridisation of e10, e15, and neonatal pancreas using a DIG-labelled Frz3 probe (green pseudo-colour in D and dark blue in E, F) counterstained with antibodies against IPF1/PDX1 (red) in D (Paper III, pp 27).

Wnt 4 is a crucial modulator of renal development and mice with targeted

deletion of Wnt4 display agenic kidneys and complete lack of nephrons

(Stark et al, 1994). Wnt 4 comprises also one of the key genes involved in

mammalian sex-determination. Female Wnt4-/- mice show absence of

Müllerian ducts and ectopic testosterone synthesis (Vainio et al, 1999). No

59

expression of Wnt4 was observed in the early pancreatic progenitors

(Paper III, Fig. 1A). Pancreatic Wnt4 expression was first observed at e13-

14 within the streaks of emerging endocrine cells; robust wnt4 expression

was evident also in the intestinal and stomach epithelia at these stages

(Paper III, Fig. 1B and insert in B). At e13-14 Wnt4 expression co-localized

with that of ngn3 in the proendocrine clusters (Paper III, Fig. 1B) whereas

Wnt4 expression at later stages became restricted to the islets of neonatal

mice with preferential high expression in the α-cells (Paper III, Fig. 1C and

data not shown).

Targeted deletion of Wnt7b leads to respiratory failure and lung hypoplasia

in mice due to defects in early mesenchymal proliferation suggesting a key

role for Wnt7b during lung development. In addition, homozygous Wnt7b

mutant mice die at midgestation stages as a result of placental

abnormalities (Parr et al, 2001) demonstrating that Wnt7b signalling is

central to the early stages of placental development in mammals. Wnt7b

expression was first detected in the pancreatic ductal epithelium at stage

13 and onwards with little or no expression in the pancreas at the neonatal

stage (data not shown).

Frz3 has been shown to be essential for anterior-posterior guidance of

commissural neurons (Lyuksyutova et al, 2003). Wang and co-workers

(2002) generated Frz3-/- mice displaying defects in fiber tracts in the rostral

CNS (Wang et al, 2002). Frz3 expression was detected in IPF1/PDX1+

progenitors in the early pancreatic buds (Paper III, Fig. 1D). The expression

was maintained in the developing pancreatic epithelium until e15 (Paper III,

Fig. 1E-F) but appeared progressively reduced at later stages (data not

shown). The presence of Frz3 in the early pancreatic progenitor cells is in

agreement with a role for this receptor in mediating Wnt signals that

promote subsequent growth and morphogenesis of the early embryonic

pancreas.

60

The Ipf1/Frz8CRD mice show reduced pancreatic mass due to

decreased epithelial pancreatic cell proliferation.

A number of independent studies have demonstrated a key role for Wnt

signalling in cell proliferation. For example, Wnt signalling is required in the

entire nervous system for expanding the progenitor cell population by

simultaneously promoting cell proliferation and blocking apoptosis and

differentiation (Zechner et al, 2003). Studies in colorectal cancer cell lines

and in transgenic mice where Dkk1, a Wnt antagonist, is ectopically

expressed have also demonstrated an essential role of Wnt signalling in the

maintenance of intestinal stem cells by promoting proliferation and blocking

differentiation (Pinto et al, 2003; van de Wetering et al, 2002; Sancho et al,

2003).

To decipher a potential role for Wnt-signalling in pancreatic growth we next

generated transgenic mice expressing the extracellular cysteine-rich

domain (CRD) domain of mouse Frz8 in the developing and adult pancreas

using the Ipf1/Pdx1 promoter which would act as a dominant negative to

block Wnt signaling in the pancreas (Figure 12).

Figure 12. Schematic representation of the effect of the transgene construct on Wnt signaling in the pancreas. To elucidate a potential role for Wnt-signalling in pancreatic

61

growth. Wnts bind to Frz receptors via the CRD domain (left), which is highly conserved between different Frz members. Hence, the CRD domain alone functions as a secreted antagonist of Wnt signalling (right) by sequestering the Wnts that are present preventing them from binding to the endogenous Frz receptors and activate downstream cascades (Hsieh et al, 1999; Lyons et al, 2004).

Ipf1/Frz8CRD neonates were born alive, appeared indistinguishable from

their littermates but presented with a drastically reduced pancreas (Paper

III, Fig. 2A). The net weight of the transgenic pancreas was merely 25% of

that of the wt (Paper III, Fig. 2B). Together these data indicate that Wnt

signalling is critical for pancreatic growth.

Next, we set out to investigate whether the reduction in pancreatic mass

displayed by the Ipf1/Frz8CRD mice reflected perturbations in initial

pancreatic specification, proliferation, apoptosis or premature

differentiation. Defect pancreatic specification would reflect a role for Wnt

signalling in controlling and/or mediating the molecular cues that define

and/or induce the pancreatic domain along the primitive gut endoderm. If

Wnt signalling is involved in the early specification of the pancreatic

program, the phenotype would be evident already at e9-e10. Using

wholemount immunohistochemistry on transgenic and wt e10 embryos we

compared the size of the pancreas at this stage (Paper III, Fig. 3A). No

obvious difference was apparent in the size of the dorsal and ventral buds

of e10 transgenic versus wild type embryos. Secondly, to exclude that the

reduced pancreatic size was the consequence of premature differentiation,

we examined the expression of differentiation markers in e13 pancreases.

Activated, intracellular Notch blocks pancreatic cell differentiation by

repressing the expression of the proendocrine gene ngn3 (See Introduction

and Apelqvist et al, 1999; Jensen et al, 2000). In the absence of activated

Notch, pancreatic progenitors differentiate prematurely into the endocrine

lineage at the expense of pancreatic progenitor cell expansion (Apelqvist et

al, 1999). No increase in the expression of the proendocrine marker ngn3

62

(Paper III, Fig. 3A) or the endocrine marker ISL1 (data not shown) was

observed and the expression of Notch1 was indistinguishable from that

observed in wt embryos of the same stage (Paper III, Fig. 3A). Thirdly, to

investigate whether an increase in apoptosis could explain the reduced

pancreatic mass observed in the Ipf1/Frz8CRD mice, transgenic and wt

pancreata at stages e13-17 were stained for the apoptotic marker Caspase

3. There was no difference in the apoptotic index between transgenic and

wt mice. Finally, we performed immunohistochemistry using the mitotic

marker phosphohistone-H3 and counted the number of phosphohistone-

H3-positive cells over DAPI, i.e. total cell number. We detected a 25%

reduction in the proliferating cells in the pancreas of e11 transgenic

embryos and an even greater reduction, by 50%, at e13 (Paper III, Fig. 3B).

At e15, when pancreatic progenitor cell proliferation is declining also in wt

mice, the difference in the proliferation rate was merely 20% (Paper III, Fig.

3B). Taken together, these data provide evidence that the reduced size in

Ipf1/Frz8CRD mice is the consequence of decreased pancreatic cell

proliferation rather than perturbed specification, premature differentiation or

increased apoptosis.

FGF, Wnts and EGFs are known to act in synergy to promote proliferation

in a variety of systems (Reya et al, 2003; Lee and Jessell, 1999; Jernvall

and Thesleff, 2000; Otto and Rao, 2004). Gross morphological analysis of

Ipf1/Frz8CRD neonates revealed pancreatic hypoplasia, as early as at e14.

Fgf10-/- mice also display severe pancreatic hypoplasia indicating of a role

for both signalling pathways in pancreatic growth. However, there is a

distinct difference between the Ipf1/Frz8CRD and Fgf10-/- mouse models.

The Fgf10-/- mice show complete pancreatic agenesis and severely

reduced expression of IPF1/PDX1 in early pancreatic progenitors (Bhushan

et al, 2001). The pancreatic growth arrest observed in the Fgf10-/- mice in

fact resembles that in Ipf1/Pdx1-/- (Jonsson et al, 1994; Offield et al, 1996)

mice. In contrast, IPF1/PDX1 expression is not perturbed in the

63

Ipf1/Frz8CRD mice, and proliferation occurs albeit at a clearly reduced rate.

Combining these data with those from the Ipf1/Fgf10 mice (section 5.1.2),

Wnt signalling may be acting synergistically with FGF10 signalling to

ensure normal proliferation rate and proper pancreatic growth; FGF10 in

turn, interacts -directly or indirectly- with Notch signalling (perhaps by

controlling Sel-1l expression) to regulate differentiation of pancreatic

progenitor cells (Figure 13).

Figure 13. Model for a possible crosstalk between FGF10, Wnt signaling and Notch to regulate proliferation and differentiation of early pancreatic progenitors (see text above and section 5.1.2)

The Ipf1/Frz8CRD mice show normal endocrine and exocrine

function.

To analyse in detail the differentiated state of the pancreas in Ipf1/Frz8CRD neonates, we examined the expression of the hormones insulin and

glucagon, transcription factors ISL-1, IPF1/PDX1 and carboxypeptidase

(CPA) enzyme. Expression of all the markers mentioned above was

apparently normal in the pancreas of the Ipf1/Frz8CRD mice (Paper III, Fig.

4A-H) except for IPF1/PDX1 whose immunoreactivity appeared slightly

increased in β-cells of the transgenic mice compared to that of wt

littermates suggesting that levels of Wnt signalling might regulate directly or

indirectly, IPF1/PDX1 expression or that the β-cells compensate for the

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reduced pancreatic mass by improving β-cell function (Paper III, Fig. 4C-D).

The islets of the Ipf1/Frz8CRD mice appeared smaller in size and showed a

typical organisation with the β-cells residing in the core of the islet and the

α-cells in the periphery (Paper III, Fig. 4A-B). The transgenic mice also

presented with fully developed acinar structures and CPA expression in

transgenic pancreas appeared indistinguishable from that of the controls

(Paper III, Fig. 4G-H). Collectively these data show that although

attenuated Wnt signalling perturbs pancreatic epithelial cell proliferation it

does not affect either endocrine or exocrine cell differentiation.

The Ipf1/Frz8CRD mice are normoglycaemic due to the presence of

putative compensatory mechanisms.

Numerous teams have reported on compensatory adaptations by adult β-

cells in response to variations in insulin demands (Bernard-Kargar and

Ktorza, 2001). A 40-60% reduction in β-cell mass in rodents does not result

in abnormal glucose homeostasis; 85-95% pancreatectomy is required to

induce hyperglycaemia in these rodents (Bonner-Weir, 1988; Leahy et al,

1988). Mice with a targeted disruption of the insulin receptor substrate 1

(IRS-1) develop insulin resistance but maintain normoglycaemia via a

compensatory increase in β-cell mass (Tamemoto et al, 1994; Terauchi et

al, 1997). Rats infused with glucose or lipids where shown to achieve

maintenance of normoglycaemia by dramatically increasing their insulin

secretory response paralleled by enhanced β-cell proliferation 1-2 days

post-infusion leading to subsequent increase in β-cell mass (Steil et al,

2001). Recently, chemical reduction of β-cell mass in minipigs, which

provokes glucose intolerance, resulted in an increased insulin response

from the residual β-cell population during a mixed meal tolerance test

(Larsen et al, 2004).

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Despite the drastic reduction in size, the transgenic mice did not display

signs of either hyperglycaemia or diabetes and showed a normal glucose

tolerance when challenged with exogenous glucose (Paper III, Fig. 5A).

Serum insulin levels in response to glucose challenge were normal in non-

fasted Ipf1/Frz8CRD mice with a normal first and second phase insulin

release (Paper III, Fig. 5B). In addition, determination of the relative insulin

content in total pancreatic extracts (ng insulin/mg pancreatic protein) from

transgenic and wt mice revealed a ~50% increase in relative insulin content

in transgenic mice compared to age-matched controls (Paper III, Fig. 5C).

These results suggest that insulin biosynthesis and/or the relative number

of the insulin-positive cells is increased in the transgenic mice. To clarify

the latter, we quantified numbers of differentiated endocrine cell types

against total endocrine cell count and against total pancreatic area in

neonatal pancreas (Paper III, Fig. 5D). There was an ∼50% reduction in the

absolute numbers of endocrine cells (i.e. ISL1-positive cells), however,

there were ∼30% more endocrine cells per pancreatic area and no

difference was observed in the proportion of β- versus α- cells over total

endocrine cells (Paper III, Fig. 5D).

Apart from the relative increase in β-cells, improved insulin biosynthesis

and/or enhanced insulin secretion could also contribute to the 50%

increase in relative total insulin content in the transgenic mice versus wt

controls. To explore this, we calculated the total relative insulin content and

total insulin secretion per unit of β-cell mass, i.e. per individual β-cell, and

observed a 4- and a 2-fold increase respectively, when compared to control

mice (Paper III, Fig. 5E). In agreement with an enhanced insulin

biosynthesis, the expression of prohormone convertase, 1/3 (PC1/3) was

increased 3-fold in islets of Ipf1/Frz8CRD mice compared to wt littermates.

Further hints towards the presence of putative compensatory mechanisms

adopted by the pancreas of the Ipf1/Frz8CRD mice came from quantitative

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RT-PCR analysis of the expression the key β-cell factor Ipf1/Pdx1

expression that showed a ∼2.5-fold increase, consistent with the increased

protein levels detected by immunohistochemistry (Paper III, Fig. 5F). These

data indicate that the improved β-cell function in Ipf1/Frz8CRD mice is

mediated, at least in part, by the increased expression of Ipf1/Pdx1, which

in turn has been suggested to transactivate several β-cell genes including

insulin, Glut2 and PC1/3. The maintained or even improved β-cell function

and glucose homeostasis observed in the Ipf1/Frz8CRD mice suggests that

attenuation of Wnt signalling in adult β-cells does not affect glucose

sensing. In summary, our data indicate that the normoglycaemia observed

in Ipf1/Frz8CRD mice most likely is achieved by combination of

compensatory mechanism that include increased relative endocrine cell

number, enhanced insulin biosynthesis and insulin secretion. The

normoglycaemia displayed by Ipf1/Frz8CRD mice suggest that Wnt-

signalling is not critical for normal glucose metabolism and insulin secretion

thus contradicting data obtained from the LRP5 -/- mice that displayed

impaired glucose tolerance and GSIS when glucose challenged (Fujino et

al, 2003). The difference in glucose tolerance and insulin secretion in the

LRP5-/- mice was, however, age-dependent and developed first after 6

months leaving open the possibility that the β-cell defects in LRP5-/- mice

are secondary rather than primary.

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6.EPILOGUE

Pancreatic development and prospects for therapy

“Our doubts are traitors, and make us lose the good we oft might win By Fearing to attempt…”

(William Shakespeare, Measure for Measure)

Today, pancreatic islet cell transplantation is an established procedure to

restore normal β-cell numbers and normoglycaemia in Type I diabetic

patients (Shapiro et al, 2000) and potentially in patients with Type II.

However, the use of this therapy has limitations due to the shortage of

transplantable material. For this reason, a hope of investigators studying

pancreas development has been to isolate a stem cell that could be

induced to generate new β-cells. The problems: where to find such stem

cells and how to control their differentiation.

Histological studies of endocrine development suggested that the early

epithelium might comprise a stem cell population (Pictet and Rutter, 1972).

But do these putative stem cells persist at later stages of pancreas

development? Studies in rats treated with the β-cell toxin streptozotocin

showed that the ability of pancreas to regenerate diminishes postnatally. In

addition, experiments including partial pancreatectomy (Bonner-Weir et al,

1993) and duct ligation (Wang et al, 1995) show that the adult pancreas

retains some capacity to produce new β-cells through a process called

neogenesis. It has been suggested that the pancreatic ducts might serve a

function analogous to that of the early epithelium. Hence, that raises two

possibilities: either stem cells reside within the ducts or that the entire

ductal epithelium has the potential to acquire stem cell properties. Studies

of pancreatic ductal epithelium present with several limitations, the main

being the lack of specific markers for ducts in adult pancreata. Many

markers used are also expressed in the acini and endocrine cells, leading

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to the conclusion that the duct-like structure generated in the regenerating

pancreas as a result of neogenesis might in fact not be related to the ducts

of the intact organ. Lineage tracing work (Gu et al, 2002) has suggested

that adult ducts arise from a subpopulation of IPF1/PDX1-expressing

progenitor cells. Hence, a bone-fide pancreatic stem cell for β-cell

regeneration remains elusive. A genetic-marking study in mice casts doubt

on the existence of any β-cell progenitor cells and suggests that β-cells

regenerate only by proliferation of existing β-cells (Dor et al, 2004)

Alternatively, stem cells in the adult pancreas might arise via the de-

differentiation of existing cell types. One potential source, in addition to

ductal cells, is acinar cells. Several mouse models have showed evidence

of acinar-to-duct transdifferentiation such as the mice expression interferon-

gamma (IFN-γ) in β-cells (Gu et al, 1994) and those expressing TGFα in the

exocrine pancreas (Song et al, 1999). However, these studies rely on

histological rather than genetic evidence and the possibility remains that

the embryonic epithelium found in the pancreas of these mice is a remnant

of the embryonic pancreas rather than the result of transdifferentiation.

Finally, given widespread current reports of adult stem cell plasticity, other

sources of stem cells, outside the pancreas itself, have not been ruled out.

It has been suggested that new β-cells could be generated from the bone

marrow in response to injury (Blau et al, 2001). Bone marrow cells can

differentiate in vitro under controlled conditions into insulin–expressing cells

(Jiang et al, 2002; Jahr and Bretzel, 2003). Such cells, transplanted under

the kidney capsule of diabetic rodents, alleviate hyperglycaemia but upon

removal of the grafted kidney, diabetes resumes (Oh et al, 2004).

Nevertheless, transdifferentiation has spurred intense controversy. Cell

fusion, rather than transdifferentiation, has been suggested as the putative

mechanism that allows bone marrow-derived cells to acquire an

extramedullary phenotype (Kofman et al, 2004; Thiese and Wilmut, 2003).

69

Indeed, some groups have not observed any transdifferentiation or bone-

marrow derivation of intra-islet endothelial cells in diabetic mice (Thiese

and Wilmut, 2003; Wagers et al, 2002; Lechner et al, 2004).

Paradoxically, even though the existence of a pancreatic stem cell has not

been proven to date, numerous laboratories have reported successful β-cell

neogenesis in various culture systems, including fetal human pancreas

(Beattie et al, 1996), adult ducts (Bonner-Weir, 2000; Ramiya et al, 2000),

adult islets (Zulewski et al, 2001) and embryonic stem cells (Lumelsky et al,

2001; Soria 2001). However, the cells obtained in these studies expressed

very low levels of insulin and lacked expression of IPF1/PDX1 possible

representing neuronal cells which are known to be capable of producing

low levels of insulin (Edlund 2002). Furthermore, Rajagopal and associates

have shown that caution should be applied in such attempts since cells

tend to absorb insulin from the culturing medium rather than producing it

themselves (Rajagopal et al, 2003). Therefore, rigorous analysis of marker

gene expression in combination with carefully controlled functional

assessment, both in vitro and in vivo, of in vitro-derived insulin-producing

cells must be undertaken to assure that true β-cells are obtained. For that,

studies of pancreatic development in the future will prove invaluable in

identifying all those extrinsic and intrinsic factors as well as the

mechanisms required to ensure proper generation of fully functional β-cells

so that one day, cell-based therapies for diabetes will be fact, not fiction!

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7. CONCLUSIONS

FGFR2 is expressed in undifferentiated pancreatic progenitors from

e9.5 to e13 but becomes restricted to differentiated β-cells at later

stages.

Overexpression of FGF10 in mice leads to pancreatic hyperplasia

due to sustained pancreatic epithelial cell proliferation.

Overexpression of FGF10 in mice impairs endocrine and exocrine

differentiation with subsequent near lack of differentiated pancreatic

cell types. Instead, the pancreas of Ipf1/Fgf10 mice consists mainly

of proliferating progenitors that co-express the progenitor cell

markers IPF1/PDX1, Nkx2.2, NKx6.1 and Ptf1α/p48.

The impaired pancreatic cell differentiation observed in Ipf1/Fgf10

mice is the consequence of activated Notch. Notch signalling ‘locks’

pancreatic epithelial cells in an undifferentiated, progenitor-like state

with maintained proliferative capacity.

Persistent FGF activation leads to diabetes in mice due to impaired

glucose, sensing, glucose and glucose metabolism, which in turn

leads to perturbed insulin secretion.

Excess FGF2 signalling results in increased expression of the FGF

inhibitors Spry-2 and Pyst1/MKP3 implicating the operation of

negative feedback mechanisms in the β-cells to regulate FGF

activity. We have previously shown that perturbed FGFR1c

signalling leads to the development of diabetes, demonstrating a

71

key role for FGF signalling in the maintenance of β-cell function and

glucose homeostasis. The data presented here provide evidence

that over-stimulation of FGF signalling is as deleterious for β-cell

function as attenuation of FGF signalling.

Wnt4, Wnt7b and Frizzled 3 are expressed in the mouse pancreas.

Attenuation of Wnt signaling in mice expressing a dominant

negative mouse Frizzled 8 form leads to severe pancreatic

hypoplasia due to decreased pancreatic epithelial cell proliferation.

Perturbed Wnt signaling does not affect endocrine and exocrine

differentiation.

Despite the drastic reduction in pancreatic mass, the Ipf1/Frz8CRD

mice remain normoglycaemic due to compensatory adaptations

such as increased relative number of endocrine cells together with

enhanced glucose sensing, insulin biosynthesis and insulin

secretion. Hence, Wnt signaling is required for pancreatic growth

but not pancreatic function.

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8. ACKNOWLEDGEMENTS This thesis would not have been possible without the love, help, support and encouragement of the following people: My supervisor, Helena Edlund, for GREAT scientific discussions and financial back up, GREAT dinners and even GREATER fights! Helena, your passion for discovery and knowledge is truly an inspiration! All members of Helena’s group (PhD students, Post-Docs and technical staff) for a very prolific collaboration and especially Pär ‘Tin Tin’ Steneberg, Elisabet ‘Bettan’ Pålsson and Joan Goulley (Joan, don’t EVER change ☺!!) for that ‘extra something’ called genuine sense of humour to spice the day! Also, former celebrity members Alan Hart (who co-authored 2 of my papers) and Natalie Baeza, my mentors, deserve huge THANKS!! Our neighbours (Leif Carlsson, Thomas Edlund and Ulf Ahlgren groups) are also acknowledged for lab tips, discussions and parties. Projects students Anna Johansson, Elisabeth Berglöf and Jessica Berqvist are on that list too for being så jätte duktiga, snälla och gulliga! Jessica you are the LOUDEST Swede I have ever met, when you visit me in Greece next summer you will fit right in!! Animal technicians Ingela Berglund-Dahl, Carola Granberg and Marie Engman for taking care of my animals (including my dog, oh, yes that’s you Ingela!) and helping me out with various tests. Ulla-Britt Backman and Kerstin Falk over at Umeå Transgene Facility for ‘animating’ our scientific hypotheses! Kristina Strindlund, our department secretary, who’s helped me solve administrative riddles numerous times and PA Lundström for taking care of almost everything else! My dance instructors Miguel Jaramillo and Jocelyn Vásquez for being such good friends and for chronically and terminally infecting me with the salsa virus (the LA strain!). Amigos, gracias por haberme abierto nuevos senderos en la busqueda de mí creatividad, feminidad y por supuesto…formas de seducir…;)! Flamenco guru, the one and only Sofia Lindgren for introducing me to the intricate world of flamenco which I am sure I’m bound to get lost in. Ole! Butterfly-enthusiast, IKSU-detester, major Björken sponsor and fellow salsero…Thomas ‘hardcore’ ‘extreme’ Werner! Thomas you are one of a kind! Meeting you at Havanna that Wednesday marked the beginning of our friendship! Thank you for being SO understanding…! I’m going to miss having you around! EEHC!!! X Sivan ‘Udi’ Cohen, princess of Amsterdam, you are simply THE BEST! You are my sunshine, I love you girl. Mariusz Lewicki who’s had a hard time staying away from raunchy latinas (honey, I don’t blame you!). Mariusz, trekking with you has permanently changed my views on the inverse relation between men and endurance…! Can’t wait to see you again my friend when you are in London!

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At the UK front, my old friend Nick ‘pumkin’ Ricioppo for listening when noone else around me would. We’ve been through a great deal together; I am so lucky to have you in my life. Looking forward to seeing more of you next year☺! My long-term friends from Greece who I think the world of, Panos Karayannis and Apostolia Pimenidou, although we’ve spent the last 5 years apart, our friendship has proved to be stronger than ever before. You’ve been there from day one. I love you guys! XXX. My good friend from our time in Hull, Dimitra Papefthimiou, who I had not seen in 4 years but I met accidentally on the plane to Amsterdam 3 years ago and found out she was moving to Stockholm! Dimitra, you are exceptional; Thank you for your support, it means a lot! Good luck with the new project! ☺ My insect genius/king of power naps (my fault…sorry!) Johannes Bergsten who came into my life towards the end of my studies but who, nevertheless, has made this process a LOT sweeter…I adore your passion, intelligence and good heart. To be continued baby…in the UK☺!! Last but absolutely NOT least, my parents and baby (relatively) sister (who followed the example of her big sister and became citizen of the world!) for always being an inexhaustible source of love and comfort, no matter what! This book is dedicated to you. Σας αγαπω πολυ! XX

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