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Fgf9 signalling stimulates Spred and Sprouty expression in embryonic mouse pancreas mesenchyme

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Fgf9 signalling stimulates Spred and Sprouty expression in embryonic mouse pancreas mesenchyme

SYLVESTERSEN, Kathrine B, et al.

Abstract

Epithelial-mesenchymal interactions are critical for normal pancreas development. Fibroblast growth factor (Fgf)-10 is expressed in the pancreatic mesenchyme and its signalling is required for normal growth and regulation of gene expression in the pancreatic epithelium.

However, little is known about putative Fgf signalling to the mesenchyme. Here we have examined the embryonic pancreas expression of differentially spliced Fgf receptor isoforms and their targets; the Sprouty (Spry) and Spred family genes which are induced by Fgf signalling. Using qPCR to quantify mRNA levels in microdissected pancreatic epithelium and mesenchyme as well as in FACS isolated Pdx1-GFP(+) and -GFP(-) cell populations we demonstrate that several members of the Spred and Sprouty families are expressed in embryonic mouse pancreas and find Spred1 and -2 as well as Spry2 and -4 to be predominantly expressed in pancreatic mesenchyme. Using embryonic pancreas explant cultures we demonstrate that Spred1/2 and Spry2/4 expression is regulated by Fgf receptor signalling and is increased by treatment with Fgf9, but not by Fgf7 or Fgf10. We extend [...]

SYLVESTERSEN, Kathrine B, et al . Fgf9 signalling stimulates Spred and Sprouty expression in embryonic mouse pancreas mesenchyme. Gene Expression Patterns , 2011, vol. 11, no. 1-2, p. 105-11

DOI : 10.1016/j.gep.2010.10.001 PMID : 20934536

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http://archive-ouverte.unige.ch/unige:25118

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Fgf9 signalling stimulates Spred and Sprouty expression in embryonic mouse pancreas mesenchyme

Kathrine B. Sylvestersen

a

, Pedro L. Herrera

b

, Palle Serup

a,

, Claude Rescan

a

aHagedorn Research Institute, Department of Developmental Biology, Gentofte, Denmark

bUniversity of Geneva, Department of Cell Physiology and Metabolism, Geneva, Switzerland

a r t i c l e i n f o

Article history:

Received 26 May 2010

Received in revised form 30 September 2010

Accepted 2 October 2010 Available online 8 October 2010

Keywords:

Pancreas Sprouty Spred FGFR Mesenchyme Epithelium

a b s t r a c t

Epithelial–mesenchymal interactions are critical for normal pancreas development. Fibroblast growth factor (Fgf)-10 is expressed in the pancreatic mesenchyme and its signalling is required for normal growth and regulation of gene expression in the pancreatic epithelium. However, little is known about putative Fgf signalling to the mesenchyme. Here we have examined the embryonic pancreas expression of differentially spliced Fgf receptor isoforms and their targets; theSprouty(Spry) andSpredfamily genes which are induced by Fgf signalling. Using qPCR to quantify mRNA levels in microdissected pancreatic epithelium and mesenchyme as well as in FACS isolated Pdx1-GFP+and -GFPcell populations we dem- onstrate that several members of theSpredandSproutyfamilies are expressed in embryonic mouse pan- creas and findSpred1 and-2as well as Spry2and-4to be predominantly expressed in pancreatic mesenchyme. Using embryonic pancreas explant cultures we demonstrate thatSpred1/2andSpry2/4 expression is regulated by Fgf receptor signalling and is increased by treatment with Fgf9, but not by Fgf7 or Fgf10. We extend previous work showing thatFgf9is expressed in pancreatic mesenchyme, and since Fgf9 is known to activate the mesenchyme-specific ‘‘c”-splice forms of Fgf receptors, while Fgf7 and -10 both activate the epithelium-specific ‘‘b”-splice forms of Fgf receptors, these results suggest that Fgf signalling is active in the pancreatic mesenchyme, where expression ofSpred1/2andSpry2/4 appear downstream of Fgf9 signalling.

Ó2010 Elsevier B.V. All rights reserved.

Epitheliomesenchymal interactions are important for the devel- opment of many organs, including the pancreas (Golosow and Grobstein, 1962). The epithelial component of the pancreas is de- rived from the posterior foregut endoderm which upon commit- ment to a pancreatic fate expresses the homeobox genePdx1at embryonic day (E) 8.5 in the mouse. At this early stage (8 somites) and until the 13-somite stage, the prospective pancreatic endo- derm is in close contact with the neural plate and the notochord (Wessells and Cohen, 1967), and the notochord was later shown to provide a permissive signal to the endoderm which is required for pancreas development (Hebrok et al., 1998; Kim et al., 1997).

As the notochord separates from the endoderm around the 13-so- mite stage, the dorsal aorta becomes closely associated with the dorsal endoderm from a level anterior to the liver anlage to well back on the developing intestine (Wessells and Cohen, 1967). At this stage, aortic endothelial cells provide signals to the pancreatic endoderm that are required for maintenance of dorsalPdx1expres- sion, induction ofPtf1aexpression in the dorsal pancreatic epithe-

lium, and differentiation of the earliest appearing endocrine cells (Lammert et al., 2001; Yoshitomi and Zaret, 2004). The dorsal aorta remains in contact with the endoderm until the 22-somite stage and during this time mesodermal cells gradually accumulate adja- cent to the dorso-lateral endoderm. Thereafter, further accumula- tion of splanchnic mesodermal cells between the dorsal tip of the pancreas and the aorta forces the latter away from the pancreas endoderm (Wessells and Cohen, 1967). At this stage the accumu- lating pancreatic mesenchyme produces Fibroblast growth factor (Fgf) 10 which signals via epithelial FGF receptor-2 IIIb (FGFR2b) to maintain epithelial proliferation and Pdx1 expression and to suppress premature endocrine differentiation of the pancreatic epithelium (Bhushan et al., 2001; Elghazi et al., 2002; Miralles et al., 1999). Fgfs are involved in reciprocal signalling between the epithelial and mesenchymal components of many developing organ systems. In the developing lung and cecum, Fgf9 is expressed in the epithelium and signals through FGFR1c and -2c to the mes- enchyme where it regulates mesenchymal proliferation and in- duces or maintains mesenchymal Fgf10 expression which then signals back to the epithelium (Colvin et al., 2001; Zhang et al., 2006). Fgfs bind to and activate four receptor tyrosine kinases (FGFRs) to regulate intracellular signalling controlling cell prolifer- ation, differentiation and migration. FGFR1, FGFR2 and FGFR3 are

1567-133X/$ - see front matterÓ2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.gep.2010.10.001

Corresponding author. Address: Hagedorn Research Institute, Niels Steensensvej 6, DK 2820 Gentofte, Denmark. Tel.: +45 4443 9822; fax: +45 4443 8000.

E-mail address:pas@hagedorn.dk(P. Serup).

Contents lists available atScienceDirect

Gene Expression Patterns

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / g e p

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alternatively spliced at exon 3, encoding the D3 domain region of the receptor, giving rise to one of two alternative ‘‘b” or

‘‘c”-isoforms (Itoh and Ornitz, 2004). The ‘‘b” isoforms are typically expressed in epithelia, while the ‘‘c” isoforms are commonly found in mesenchymal tissues, forming the basis for specificity during reciprocal epitheliomesenchymal signalling (Ornitz and Itoh, 2001; Ornitz et al., 1996; Zhang et al., 2006).

As mentioned above, numerous studies have documented the role of Fgf7 and 10 in the regulation of the pancreatic epithelium, but little is known about Fgf signalling to the pancreas mesen- chyme. In zebrafish, Fgf24 signalling from pancreatic endoderm to lateral plate mesoderm is required for ventral pancreas develop- ment (Manfroid et al., 2007), but as Fgf24 is not conserved in mam- mals it is unclear if and how mesenchymal Fgf signalling is achieved in these species. To begin to understand Fgf signalling in pancreas mesenchyme we analyzed the expression of FGFR iso- forms and downstream target genes of FGFR signalling from the Sprouty- andSpred-families in isolated pancreatic epithelial and mesenchymal tissue preparations. We demonstrate epithelium- specific expression of FGFR1b, -2b, and -4 and mesenchymal- specific expression of FGFR1c and -2c in E11.5 and E13.5 mouse pancreas. Notably, we findSpred1and-2as well asSpry2and-4 transcripts enriched in the mesenchymal fraction, and explant studies demonstrate that pancreaticSpredandSproutyexpression

is regulated by FGFR signalling. Additionally, treatment of pancre- atic explants with Fgf2, -4, -7, -9, and -10 show that only those Fgfs that efficiently activate the FGFR ‘‘c” isoforms (Ornitz et al., 1996;

Zhang et al., 2006) can also stimulateSpredandSproutyexpression.

As we confirm and extend previous observations on expression of Fgf9in pancreatic mesenchyme, our results suggest collectively that Fgf9 signalling from the outer mesothelial lining induceSpred/

Sproutyexpression in the underlying pancreatic mesenchyme.

1. Results

1.1. Embryonic mouse pancreas mesenchyme express FGF receptor IIIc splice forms and Fgf9

To begin to understand the potential for mesenchymal Fgf signal- ling we first analyzed the expression of FGFR splice forms and candi- date Fgf ligands in embryonic mouse pancreas epithelium and mesenchyme by qPCR. We separated E11.5 dorsal pancreatic epithe- lia from their surrounding mesenchyme and isolated RNA from both fractions, which was subsequently processed for qPCR analysis. To determine the purity of each fraction we analyzed the expression of epithelium-specific markers (Cdh1 (E-cadherin),glucagon, and Foxa2) or of mesenchymal markers (Hgf,Vim(vimentin), andFlk1).

As expected, we found mRNA for known epithelial markers to be

Fig. 1.Expression of FGFR splice forms and selected Fgfs in the E11.5 mouse embryonic pancreas. (A) qPCR analysis of known epithelial and mesenchymal markers in isolated epithelium and mesenchyme. Vimentin, Flk1 and HGF were used as mesenchymal markers and E-cadherin, Foxa2, Glucagon and Pdx1 were used as epithelial markers. (B).

qPCR analysis for FGFR splice form expression in isolated epithelium and mesenchyme. (C) qPCR analysis of Fgf9 and Fgf10 expression in isolated epithelium and mesenchyme. Note that both are found enriched in the mesenchyme. Data represent mean ± SEM (n= 3); *p< 0.05, **p< 0.01.

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abundant in the epithelial fraction but scarce in the mesenchymal fraction (Fig. 1A), suggesting that our mesenchymal fraction is rela- tively pure. Conversely, we found known mesenchymal markers to be abundant in the mesenchymal fraction as expected, but also de- tected lower levels of these in the epithelial fraction, indicating that the latter is contaminated by mesenchymal cells (Fig. 1A). We next examined the expression of the different FGFR splice forms in the epithelial and mesenchymal fractions. As expected we found mRNA for the known epithelial receptor isoformsFGFR2bandFGFR4(Elg- hazi et al., 2002) to be enriched in our epithelial fraction, while other putative epithelial specific isoforms (FGFR1band-3b) were only ex- pressed at low levels (Fig. 1B).FGFR1has previously been found in both pancreatic epithelium and mesenchyme (Elghazi et al., 2002) but no distinction was made between ‘‘b”- and ‘‘c”-splice forms. In the epithelial fraction we found low levels ofFGFR1b, slightly en- riched compared to the mesenchymal fraction (Fig. 1B) However, in the mesenchymal fraction we found enrichment for mRNA encod- ingFGFR1cas well as-2cand the expression levels were comparable to the epithelialFGFR2bandFGFR4(Fig. 1B), suggesting that these receptors may mediate mesenchymal Fgf signalling. Expression of FGFR3 splice forms has to our knowledge not previously been ana- lyzed in isolated epithelial and mesenchymal fractions from the pancreas. We found low levels ofFGFR3band10-fold higher levels ofFGFR3c. However, we found no enrichment of either splice form in the epithelial or mesenchymal fractions (Fig. 1B). Among the Fgf li- gands it is known that Fgf10 is expressed in the mesenchyme and signals to the epithelium (Bhushan et al., 2001; Elghazi et al., 2002; Miralles et al., 1999). In mammals, little is known about pan- creatic expression and action of Fgf ligands capable of activating the

‘‘c” splice forms of the Fgf receptors. Previous work has demon- strated thatFgf9is expressed in the epithelium of many developing organs of endodermal origin as well as in the mesothelium that sur- rounds the mesenchymal component of these organs (Colvin et al., 2001; Zhang et al., 2006). Fgf9 typically signals to the mesenchymal component through the ‘‘c” splice forms of FGFR1-3 during embry- onic development of these organs. We previously foundFgf9mRNA to be expressed in embryonic pancreas by RT-PCR (Dichmann et al., 2003) and in situ hybridization revealed that Fgf9 mRNA was expressed in E10.5 mesenchyme and mesothelium (Hecksher- Sorensen et al., 2004). We therefore analyzed expression levels of Fgf9andFgf10mRNA in our E11.5 epithelial and mesenchymal frac- tions by qPCR. As expected, we foundFgf10mRNA to be enriched in the mesenchymal fraction and similarly we foundFgf9mRNA to be enriched in this fraction (Fig. 1C). In order to perform a similar anal- ysis at E13.5, the time where beta cells begin to form in appreciable numbers, we FACS sorted GFP+and GFPcells from micro-dissected E13.5Pdx1-GFP embryonic pancreas and isolated RNA from both fractions, which was subsequently processed for qPCR analysis.

The purity of each fraction was again determined by analyzing the expression of known epithelial and mesenchymal markers (Fig. 2A). We found high levels ofglucagonmRNA in the GFP+fraction which may appear surprising. However, glucagon-producing cells arise from Pdx1+ epithelium (Herrera, 2000) and residual Pdx1 immunoreactivity can be seen in newly born glucagon-producing cells (Jensen et al., 2000). Given the long half-life of GFP it is therefore not surprising to findglucagonmRNA in the GFP+fraction.

qPCR analysis of the GFP+and GFPfractions showed thatFgf9and Fgf10transcripts are enriched in the GFPfraction as expected for mesenchymally expressed genes (Fig. 2B), and confirms the absence ofFgf9message in the pancreatic epithelium.

1.2. Spred1/2 and Sprouty2/4 expression depend on FGFR activity and is stimulated by Fgf9

Four mammalianSproutyand threeSpredgenes have been iden- tified in mammals. We performed standard RT-PCR to determine

whichSpredandSproutymembers were expressed in developing E12.5 pancreas. Transcripts for all seven genes were detected althoughSpry1,Spry3, andSpred3were expressed at a very low le- vel (Fig. 3A). qPCR experiments confirmed these expressions and indicated thatSpred1andSpry2were five times and eight times more abundant than Spred2 and Spry4, respectively (data not shown). At the protein level, the pancreatic expression was con- firmed for Spred1 and Sprouty2 and appeared to decrease as devel- opment progressed. Spred2, Sprouty3 and Sprouty4 proteins could not be detected (Fig. 3B).

In order to determine more precisely the pancreatic localization of the strongly expressed Spred and Sprouty members, we per- formed qPCR of isolated E11.5 epithelial and mesenchymal frac- tions and E13.5 Pdx1-GFP+ and -GFP fractions. As shown in Fig. 4, Spred1and Spry2transcripts were enriched in the E11.5 and E13.5 mesenchymal fractions.

The presence of FGFR ‘‘c”-isoforms and Fgf9 in the pancreatic mesenchyme, suggested that Fgf9 could regulateSproutyandSpred Fig. 2.qPCR analysis of Fgf9 and Fgf10 expression in FACS isolated GFP+and GFP cells from E13.5 Pdx1-GFP embryos. (A) qPCR analysis of known epithelial and mesenchymal markers in FACS sorted GFP+and GFPfractions. Vimentin, Flk1 and HGF were used as mesenchymal markers and E-cadherin, Foxa2, Glucagon and Pdx1 were used as epithelial markers. (B) qPCR analysis of Fgf9 and Fgf10 expression in GFP+ and GFP fractions. Both transcripts are found enriched in the GFP population. Data represent mean ± SEM (n= 3); **p< 0.01, ***p< 0.001.

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expression in this compartment. We therefore tested if different Fgfs that signal either exclusively or preferentially through the

‘‘b”- or ‘‘c”-isoforms of the FGFR (Fgf7, Fgf10 or Fgf2, Fgf4, and Fgf9, respectively) could stimulateSpred and Sproutyexpression in explants of embryonic pancreas.Spred1/2andSpry2/4expres- sion remained constant after Fgf7 and Fgf10 treatment, with the exception ofSpry4, that showed a slight decrease after FGF7 stim- ulation (Fig. 5A). In contrast, treatment with the ‘‘c”-isoform acti- vators Fgf2 and Fgf9 resulted a significant increase of Spred1/2 andSpry2/4 with the highest fold stimulation detected forSpry2 and-4. Fgf4 treatment also resulted in increasedSpry2/4expres- sion but was less potent than Fgf2 and -9.

To test if Fgf-signalling was required for pancreaticSpred1/2and Spry2/4expression we used two chemical inhibitors, SU5402 and U0126 which inhibit the kinase activity of FGFR and MEK1/2, respectively. We verified that treatment with these compounds inhibited phosphorylation of Erk1/2 in pancreatic explants (Fig 5B). Importantly, the basal Erk1/2 phosphorylation we detected

in absence of exogenous Fgf was also reduced by both inhibitors.

We then tested if the Fgf2 stimulatedSpred1/2andSpry2/4expres- sion was blocked by these inhibitors. Indeed, the presence of SU5402 completely blocked Fgf2 induced Spred1/2 and Spry2/4 expression in pancreatic explants, while the MEK inhibitor U0126 partially inhibited it (Fig 5C). Importantly, unstimulated expres- sion ofSpred1/2andSpry2/4was also reduced by both inhibitors, suggesting that endogenous Spred1/2 and Spry2/4 expression is regulated by Fgf signalling.

2. Discussion

To investigate the possible involvement of Spred and Sprouty members in murine pancreas development, we undertook a PCR screen for all known members of this family. Among them,Spry2 andSpred1were the most highly expressed as measured by qPCR and the only ones detectable at the protein level. By two independent methods of separation, we found their expression to Fig. 3.Expression of Spred and Sprouty in mouse embryonic pancreas. (A) RT-PCR analysis of Spred and Sprouty genes in cDNA preparations from dissected E12.5 dorsal pancreatic buds (a), E12.5 heads (b,) and water (c). The latter two preparations were used as positive and negative controls for the PCR primers, respectively. (B) Western blot analysis of Spred and Sprouty protein expression in 40lg of E12.5, E14.5 and E16.5 embryonic mouse pancreas extract. Positive controls are lysates of CHO cells transfected with expression vectors for the respective antigen. Control lysates (10lg and 2.5lg) were loaded for Spred1 and Sprouty2, respectively. Endogenous levels of Spred2 and Sprouty4 in developing pancreas are below detection level. Spred1 and Sprouty2 are detected at E12.5 and their expressions decrease with time. Anti-Erk1/2 and anti-TFIIB was used for loading control.

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be preferentially localized to the pancreatic mesenchyme. The same pattern of expression has been described forSpredgenes in the murine lungs (Hashimoto et al., 2002), whileSprouty genes are reported to be most highly expressed in lung epithelium (Hashimoto et al., 2002; Mailleux et al., 2001). Moreover, we have shown that their expressions are increased by Fgf2 and Fgf9 in pan- creatic explant cultures. We show here that expression ofSpred andSproutyis under Fgf regulation in pancreatic explants, similar to what is observed in the lung, and this inductive effect of Fgf onSpredandSproutyexpression required a functional MAPK sig- nalling pathway as shown previously in other systems (Minowada et al., 1999; Ozaki et al., 2001).

The present study extends previous work done mainly in the rat showing that the ‘‘c” splice forms of the FGFR is mainly expressed by the pancreatic mesenchyme (Elghazi et al., 2002; Cras-Meneur and Scharfmann, 2002). Previous reports in transgenic mice have shown that mesenchymal mitotic activity is increased byPdx1pro- moter driven Fgf4 expression but not in embryos where thePdx1 promoter drives Fgf10 expression (Dichmann et al., 2003; Hart et al., 2003; Norgaard et al., 2003). These studies indicate that di- rectly or indirectly, the mesenchyme can respond to a ‘‘c”-isoform specific Fgf signal. Here we confirm this notion by showing that Fgf9 and Fgf2 both stimulate mesenchymal Spred1/2andSprou- ty2/4 expression. While Fgf2 can stimulate ‘‘b”-isoforms of the FGFR it is preferentially activating the ‘‘c”-isoforms (Zhang et al., 2006). Furthermore, the inability of Fgf7 and Fgf10, which are ago- nists of FGFR1b and -2b, to induceSpred1/2orSpry2/4expression further supports that Fgf2 and -9 inducedSpred1/2andSpry 2/4 expression is occurring via activation of the ‘‘c”-isoforms. Spred andSproutygenes are induced by a number of receptor tyrosine kinase (RTK) pathways and we cannot rule out that RTKs different from FGFRs contribute to the regulation of Spred and Sprouty

expression in the pancreas. Additional input toSpredandSprouty promoters from other RTK pathways might explain that Spred1, which we found to have the highest expression level of all the Spred and Sproutygenes in pancreatic mesenchyme, is the least responsive to treatment with Fgf9. Nevertheless, inhibition of FGFR kinase activity still attenuated Spred1 expression. Conversely, Spry4 which showed the lowest expression levels was most responsive to Fgf9 treatment. Since our expression data are based on qPCR analyses of separated epithelial and mesenchymal compo- nents we cannot accurately pinpoint the site ofFgf9expression.

Most endoderm-derived organs express Fgf9 in the epithelial (endodermal) component as well as the mesothelium surrounding the associated mesenchyme (Colvin et al., 1999) but our data re- veals that the pancreas stand out among these as an organ where Fgf9is not expressed in the epithelium. It is possible that the mes- enchymalFgf9expression we detect is in fact solely derived from the mesothelium, and that the mesenchymal cells responding to Fgf9in vivoare those closest to the mesothelium which may repre- sent in minor population of all the mesenchymal cells. Thus, treat- ment of pancreatic explants with Fgf9 may activate signalling in mesenchymal cells that have not previously been exposed to Fgf9 in vivo. The fact that we observe a strong enrichment for Fgf9message (10-fold) compared to Fgf10message (2.5-fold) in mechanically separated epithelial and mesenchymal fractions is consistent withFgf9expression being restricted to the mesothe- lium since the mesenchyme that contaminates the epithelial frac- tion is likely to be that which is in close contact with the epithelium and not the outer mesothelial lining of the mesen- chyme. Unfortunately, Fgf9in situ hybridizations failed to yield reliable signals so we cannot conclusively demonstrate this notion.

The pancreas-specific lack of endodermal Fgf9 expression is notable and similar to endodermal Shhexpression which is also specifically excluded from the pancreatic epithelium. Transgenic mis-expression ofShhin pancreatic epithelium results in transfor- mation of the pancreatic mesenchyme into intestinal mesenchyme and as a consequence of this the pancreatic epithelium fails to grow and branch normally (Apelqvist et al., 1997). It may be inter- esting to examine the consequence of forced expression of Fgf9 in the pancreatic epithelium. In conclusion, we show here that the pancreatic mesenchyme express several FGFR ‘‘c”-isoforms as well as a number ofSpredandSproutygenes. Together our data indicate that Fgf signalling is active in the mesenchymal component of the pancreas also in mammals, and contributes to the regulation of pancreaticSpredandSproutyexpression. The Fgf signalling is likely to be mediated, at least partly, by Fgf9 which is expressed in the pancreatic mesenchyme, and most likely being restricted to the outer mesothelial lining of the mesenchyme.

3. Experimental procedures 3.1. Mice

Pdx1-eGFP transgenic mice were generated by pronuclear microinjection of a 6.3 kb-long transgene, which was cloned by replacing the Cre coding region of Pdx1-Cre (Herrera, 2000) by the coding sequence of eGFP. Two different families were established from separate founder mice, displaying the same phe- notype. Only one was used in these studies.

3.2. Culture of pancreatic buds

Dorsal pancreatic rudiments were dissected from embryonic day (E)11.5 NMRI mice, and cultivated in 40

l

l hanging drops.

Culture medium was DMEM/F12 + Glutamax medium supple- mented with 1% heat-inactivated fetal calf serum (Invitrogen), Fig. 4.Mesenchymal expression of Spred and Sprouty transcripts. (A) qPCR analysis

of Spred and Sprouty gene expression in E11.5 mesenchymal and epithelial fractions obtained after manual separation. Spred1 and 2, Sprouty2 and 4 are enriched in the mesenchymal fraction. (B) qPCR analysis of Spred and Sprouty gene expression in E13.5 GFP+and GFPcell populations in E13.5 pancreas from Pdx1- GFP embryos. Spred1 and Sprouty2 are found enriched in the GFPcell population.

Data represent mean ± SEM (n= 3); *p< 0.05, **p< 0.01.

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Pen/Strep (Invitrogen), and 0.04 mg/ml gentamycin (Invitrogen) and was renewed every day. Cultures were maintained at 37°C in a humidified atmosphere of 95% air–5% CO2. All the recombinant proteins were from R&D Systems, except FGF2 (Invitrogen) and were used at 100 ng/ml. Chemical inhibitors SU5402, and U0126 (both from Calbiochem) were dissolved in DMSO and used at a fi- nal concentration of 10

l

M. Comparable DMSO (Sigma–Aldrich) concentration, i.e., 0.1% of the final volume was added in control conditions. Preparation of single-cell suspensions from dissected pancreata for fluorescence activated cell sorting (FACS).

Dissected pancreata from E13.5 Pdx1-eGFP embryos were incu- bated in a solution of 0.05% (vol/vol) trypsin–EDTA (Invitrogen) at 37°C for 5 min. The digestion was stopped by adding F10 medium containing 10% (vol/vol) FCS. The resulting suspension of single cells was washed twice with ice-cold PBS and filtered through a 70-

l

m nylon mesh. FACS of GFP-labelled cells was carried out using a FACS Aria (Becton Dickinson Biosciences, San Jose, CA).

3.3. RNA isolation, cDNA synthesis, RT-PCR and qPCR

RNA from dissected embryonic pancreas, from FACS purified cells, and from pancreatic explants was extracted with Invisorb

Spin cell RNA kit and cDNA was generated from RNA using MMLV Reverse Transcriptase (Invitrogen). PCR was carried out in a 25

l

l reaction using ReddyMix from AbGene (Epsom, UK) on a PTC200 thermal cycler (MJ research, Waltham, MA) for 32 cycles, and re- solved on a 2% agarose gel. qPCR (quantitative PCR) was performed using the standard SYBRÒ Green program and the dissociation curves were made on the Mx3005P machine (Stratagene). Quanti- fied values for each gene of interest were normalized against the input determined by the housekeeping geneglucose-6-phosphate dehydrogenase. In experiments where changes in gene expression were measured after stimulation or blockade of FGF signalling, the results are expressed as the relative expression compared to vehicle controls. Primers used were: Spry1: 50-ATT TGG CCG AGA GTT GTT TG-30 and 50-TGA GAT ACC AGG GGC AAA TC-30; Spry2:

50-GGT TTT ATT CCA CCG ATT GC-30 and 50-CTG GGT AAG GGC ATC TCT TG-30; Spry3: 50-TGG AAA CAG GAA GGG AAT TG-30and 50-CAT TGC AGA CAA AGC AAG GA-30; Spry4: 50-GCC AAC TGG AAG AGA GCA AC-30 and 50-CAC AGG AAT GTG GTG GAG TG-30; Spred1: 50-TTC ACC ACT GGA AGA TCG ATG-30 and 50-TGT CTG TAG TCT GCA TAT CGA CG-30; Spred2: 50-ACA GAA GAC GCG GAC TCC TA-30 and 50-ACA GCA CCT GCA CAT CAC TC-30; Spred3: 50- GCC CGC CAG GGG CAC TAC GTC AT-30 and 50-GGG CGT GGC Fig. 5.Spred1/2 and Sprouty2/4 expression depends on FGFR and MEK-ERK signalling pathways. (A) qPCR analysis of Spred1, Spred2, Spry2 and Spry4 expression in E11.5 dorsal pancreatic buds cultured for 3 days in presence of vehicle (BSA), Fgf2, Fgf4, Fgf7, Fgf9 or Fgf10. Values are expressed as mean ± SEM (n= 3) after normalization to the vehicle control; *p< 0.05, **p< 0.01. (B) Phosphorylation status of ERK1/2 in explants stimulated as indicated for 2 h (C) qPCR analusis of Spred1, Spred2, Spry2 and Spry4 expression in dorsal pancreatic buds cultures with or without Fgf2 for 5 days in the presence or absence of SU5402 or U0126. Values are expressed as mean ± SEM (n= 4) after normalization to the vehicle control; *p< 0.05, **p< 0.01.

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CAG TGG GAA GG-30; Glucagon: 50-AGG CTC ACA AGG CAG AAA AA-30and 50-TCA TGA CGT TTG GCA ATG TT-30; Vimentin: 50-ATT TTG CCC TTG AAG CTG CTA AC-30 and 50-TCA ACC AGA GGA AGT GAC TCC AG-30; E-cadherin: 50-GAT GTG GCT CCC ACC CTC ATG- 30 and 50-CTT CTT GAA TCG GTT GCC CCA-30; Foxa2: 50-GCT GCA GAC ACT TCC TAC TAC-30 and 50-GGA CAC AGA CAG GTG AGA CT-30; Hgf: 50-CCG AGT TGG TTA CTG CTC TCA-30and 50-CCC TGT GTA GCA CCA AGG TCC-30; Fgfr1b: 50-CTT GAC GTC GTG GAA CGA TCT-30 and 50-CAC GCA GAC TGG TTA GCT TCA C-30; Fgfr1c:

50-CTT GAC GTC GTG GAA CGA TCT-30 and 50-AGA ACG GTC AAC CAT GCA GAG-30; Fgfr2b: 50-CCC ATC CTC CAA GCT GGA CTG-30 and 50-CAG AGC CAG CAC TTC TGC ATT G-30; Fgfr2c: 50-CCC ATC CTC CAA GCT GGA CTG-30 and 50-TCT CAC AGG CGC TGG CAG AAC-30; Fgfr3b: 50-CAA GTT TGG CAG CAT CCG GCA GAC-30 and 50-TCT CAG CCA CGC CTA TGA AAT TGG TG-30; Fgfr3c: 50-GGC GCT AAC ACC ACC GAC-30and 50-TGG CAG CAC CAC CAG CCA C- 30; Fgfr4: 50-GTA CCC TCG GAC CGC GGC ACA TAC-30 and 50-GCC GAA GCT GCT GCC GTT GAT G-30; Pdx1: 50- GAA CCC GAG GAA AAC AAG AGG-30 and 50-GTT CAA CAT CAC TGC CAG CTC-30; G6DPH: 50-ATG TTA TGC AGA ACC ACC TC-30and 50-ACT TTG ACC TTC TCA TCA CG-30.

3.4. Western blotting

Explants or embryonic pancreata were harvested in reporter ly- sis buffer (Promega, Madison, WI) in the presence of Halt phospha- tase and protease inhibitor (Thermo Scientific, Waltham, MA).

Lysates of CHO-K1 transfected with full length plasmid respective sequence of Spred 1, Spred 2, Sprouty 2, Sprouty 3 or Sprouty 4 were used as positive controls. Antibodies against Spred 1, Spred 2 (both from K. Schuh, Wurzburg University, Germany, and Yoshimura, Kyushu University, Japan) Sprouty 2, Sprouty 3 (Ab- cam) Sprouty 4 (Santa Cruz), phospho ERK1/2 and pan ERK1/2 (both from Cell Signaling Technology) TFIIb (Santa Cruz) were used as primary antibodies at a 1:2000 dilution. HRP-conjugated anti- body (from Santa Cruz) was used at a 1:10000 dilution.

Acknowledgements

We thank Heidi Ingemann Jensen and Søren Refsgaard Lindskog for excellent technical support, R. Scott Heller and members of Department of Developmental Biology for critical reading of the manuscript. We want to thank K. Schuh and A. Yoshimura for shar- ing of antibodies and plasmids. This work was made possible by support from the EU 6th Framework Program (to P.S.).

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