- Email: [email protected]
FGFR1-IIIb is a putative marker of pancreatic progenitor cells
Mechanisms of Development 116 (2002) 205–208 www.elsevier.com/locate/modo
FGFR1-IIIb is a putative marker of pancreatic progenitor cells Corentin Cras-Me´neur, Raphael Scharfmann* INSERM U457, Hospital R. Debre´, 48, Boulevard Se´rurier, 75019 Paris, France Received 22 February 2002; received in revised form 18 April 2002; accepted 18 April 2002
Abstract The pancreas develops from buds that derive from the endodermal epithelium of the digestive tract. The progenitor cells that will give rise to the mature pancreatic cells reside within this epithelium. However, their exact identity remains unknown. In the present study, we searched for genes expressed by pancreatic progenitor cells. We focused our search on receptor tyrosine kinases. We found that fibroblast growth factor-IIIb (FGFR1-IIIb) expression is high in pancreatic epithelium enriched in progenitor cells. We next investigated FGFR1-IIIb expression throughout pancreatic development. At early stages of pancreas development, FGFR1-IIIb is expressed by pancreatic epithelial cells that resemble undifferentiated cells, while at later stages of development, FGFR1-IIIb expression decreases, concomitant with the expected decrease in the number of progenitor cells. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pancreas; Development; Fibroblast growth factor receptor; Progenitor cells
1. Results and discussion During the past years, different potential markers of pancreatic progenitor cells have been described such as tyrosine hydroxylase (Teitelman et al., 1993), the type 2 glucose transporter (Pang et al., 1994) or nestin (Zulewski et al., 2001). Up to now, none of these markers has been fully validated. We previously showed that when E13 rat pancreatic epithelium is kept in culture for 7 days with 1% fetal calf serum (FCS), the vast majority of the cells differentiate either into endocrine or acinar cells. When the epithelium is grown for 7 days in the presence of fibroblast growth factor (FGF-7) or endothelium growth factor (EGF), endocrine differentiation is repressed, and a large number of cells stain negative for endocrine and acinar markers. Based on retrospective analysis, these cells represent progenitor for endocrine cells (Cras-Me´neur et al., 2001; Elghazi et al., 2002). We selected candidate genes whose expression could be increased in epithelia rich in endocrine progenitor cells (uncultured E13.5 epithelium and E13.5 epithelium grown with FGF-7), when compared to epithelia depleted in progenitor cells (E13.5 epithelium grown without FGF-7). We first performed comparative polymerase chain reaction (PCR) amplification on cDNAs reverse transcribed either from E13.5 pancreatic epithelium or mesenchyme to define * Corresponding author. Tel.: 133-1400-31988; fax: 133-1404-09195. E-mail address: [email protected] (R. Scharfmann).
whether the candidate genes were expressed in the epithelial fraction. As shown in Fig. 1 ( left panel), some of the screened factors were amplified exclusively from the epithelial fraction (FGFR2-IIIb, FGFR4, FGFR1-IIIb and ErbB2). Other receptors were amplified in both the epithelial and mesenchymal fractions (FGFR1-IIIc, ErbB-1 and LIFR). We next compared the level of expression of these receptors in epithelia rich in endocrine progenitor cells (uncultured E13.5 epithelium and E13.5 epithelium grown with FGF-7) to epithelia depleted in progenitor cells (E13.5 epithelium grown without FGF-7). As shown in Fig. 1 (middle panel), the level of expression of FGFR1-IIIc, FGFR2-IIIb or LIFR did not show major variations in the different conditions analyzed. The expression of ErbB-2 decreased after 7 days in culture in either condition, while the expression of FGFR-4 was decreased in epithelia grown in the presence of FGF-7. The pattern of expression of FGFR1-IIIb was the most interesting. FGFR1-IIIb expression was enriched in uncultured epithelia or epithelia grown in the presence of FGF-7, when compared to epithelia cultured without FGF-7 (Fig. 1, middle panel). We next asked whether the increase in FGFR1-IIIb expression level also occurred in conditions where the pool of endocrine progenitor cells was expanded using EGF, known to amplify pancreatic progenitor cells (Cras-Me´neur et al., 2001). As shown in Fig. 1 (right panel), the level of expression of FGFR1-IIIb was increased in EGF-treated epithelia when compared to epithelia cultured in the absence of growth factor.
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00138-7
206
C. Cras-Me´ neur, R. Scharfmann / Mechanisms of Development 116 (2002) 205–208
Fig. 1. RT-PCR screening for candidate factors differentially expressed in epithelia enriched or poor in progenitor cells. Left panel: expression profiles of the different candidate factors in E13.5 epithelium and mesenchyme. Middle panel: expression profiles of the different candidate factors in uncultured E13.5 epithelium, epithelium cultured 7 days in control conditions or with FGF-7. Right panel: expression profile of FGFR1-IIIb in epithelia cultivated without or with EGF.
Fig. 2. Localization of the FGFR1-IIIb transcripts on sections of epithelia cultured for 7 days with FGF-7. (A) In situ hybridization was performed with a FGFR1-IIIb sense probe. (B) In situ hybridization was performed with a FGFR1-IIIb antisense probe. (C) Immunohistochemistry for insulin 1 glucagon (red) and amylase (green) on the section shown in B. (D) Reconstructed image composed of the images in shown in B and C. FGFR1-IIIb signal is shown in blue.
C. Cras-Me´ neur, R. Scharfmann / Mechanisms of Development 116 (2002) 205–208
To identify the cell types expressing FGFR1-IIIb, sections of epithelia grown with FGF-7 were hybridized for FGFR1-IIIb and then stained using anti-carboxypeptidase-A, anti-insulin and -glucagon antibodies. As shown in Fig. 2, the cells expressing FGFR1-IIIb transcripts, stain negative for acinar and endocrine markers. The same experiment was next performed on pancreatic
207
sections at different stages of development. FGFR1-IIIb transcripts were detected at E13.5, E16.5 and E18.5 but disappeared at later stages. At all stages analyzed, FGFR1-IIIb transcripts were detected only in epithelial structures. At E13.5, the vast majority of the FGFR1-IIIbexpressing cells stain negative for endocrine and acinar markers. Few cells coexpress FGFR1-IIIb and carboxypeptidase. At E16.5 and E18.5, few FGFR1-IIIb-expressing cells were detected that stain negative for endocrine and acinar markers. Later in development no FGFR1-IIIb expressing cells were detected in the pancreas (Fig. 3). In conclusion, we found that FGFR1-IIIb transcripts were enriched in epithelia grown in the presence of FGF-7 or EGF, when compared to epithelia grown in the absence of FGF-7 (or EGF), suggesting that FGFR1-IIIb represents a marker for progenitor cells. 2. Experimental procedures 2.1. Dissection and organ culture Dissections and cultures of pancreatic epithelia were performed as described previously (Cras-Me´ neur et al., 2001; Elghazi et al., 2002). 2.2. RNA extraction and comparative PCR Total RNA was extracted from pools of 30–40 pancreatic epithelia and reverse transcribed. To ensure semi-comparaTable 1 Oligonucleotides used for amplification Primer name 0
Fig. 3. Localization of the FGFR1-IIIb transcripts at different stages of the pancreatic development. Left panel: in situ hybridizations using a FGFR1IIIb antisense probe were performed on pancreatic sections from E13.5, E16.5, 18.5, 20, newborn and adult rats. Right panel: the sections first hybridized using a FGFR1-IIIb antisense probe, were next stained using anti-insulin 1 anti-glucagon antibodies (revealed in red) and anti-amylase antibody (revealed in green). Images were reconstructed and FGFR1-IIIb signal is presented in blue. Long arrows indicate cells that stain positive for FGFR1-IIIb and negative for endocrine and acinar markers. Arrowheads indicate cells that express both FGFR1-IIIb and amylase.
Insulin 5 Insulin 3 0 Glucagon-5 0 Glucagon-3 0 Amylase-5 0 Amylase-3 0 Cyclophilin-5 0 Cyclophilin-3 0 GAPDH-5 0 GAPDH-3 0 HNF-3b-5 0 HNF-3b-3 0 FGFR-1IIIc-5 0 FGFR-1IIIc-3 0 FGFR-2IIIb-5 0 FGFR-2IIIb-3 0 ErbB-2-5 0 ErbB-2-3 0 ErbB-1-5 0 ErbB-1-3 0 LIFR-5 0 LIFR-3 0 FGFR-4-5 0 FGFR-4-3 0 FGFR-1IIIb-5 0 FGFR-1IIIb-3 0
Primer sequence 5 0 -CCTAAGTGACCAGCTACA-3 0 5 0 -GTAGTTCTCCAGTTGGTA-3 0 5 0 -CTCAAGACACGGAGGAGAAC-3 0 5 0 -TTCACCAGCCAAGCAATGAAT-3 0 5 0 -TCGAACCAAGGTGGCTGACTA-3 0 5 0 -GGGCTCTGTCAGTAGGCACAA-3 0 5 0 -CAGGTCCTGGCATCTTGTCC-3 0 5 0 -TTGCTGGTCTTGCCATTCCT-3 0 5 0 -GTGATGCTGGTGCTGAGTATG-3 0 5 0 -AGTTGTCATGGATGACCTTGG-3 0 5 0 -TTGCTCCCTACGCCAATATGA-3 0 5 0 -GGGCACCTTGAGAAAGCTGTT-3 0 5 0 -TGGGAGCATTAACCACACCTACC-3 0 5 0 -GCACCTCCATTTCCTTGTCG-3 0 5 0 -GAGCACCGTACTGGACCAACAC-3 0 5 0 -TGGTAGGTGTGGTTGATGGACC-3 0 5 0 -AACTTGGAGCTTACCTACGTGCC-3 0 5 0 -TATCGACAGGAGCCAGTTGGTTA-3 0 5 0 -AAACTTGGAAATCACCTATGTGCAA-3 0 5 0 -CTCAGAAAGACATCTTGGACGATGT-3 0 5 0 -TACTGCCCACCCATCATTGAG-3 0 5 0 -TCTGTGGACCTTGGGGAATCT-3 0 5 0 -AAGGATTTGGCAGACCTGATC-3 0 5 0 -TGCACTTCCGAGACTCCAGATAC-3 0 5 0 -CGGGGATTAATAGCTCGGATG-3 0 5 0 -GCACAGGTCTGGTGACAGTGA-3 0
208
C. Cras-Me´ neur, R. Scharfmann / Mechanisms of Development 116 (2002) 205–208
tive amplification within non-saturating conditions, amplifications were performed on series of two-fold dilutions of the cDNAs as described (Basmaciogullari et al., 2000). Negative controls (without reverse transcription) did not show any amplification (data not shown). The oligonucleotides used for amplification are reported in Table 1.
hybridization and B. Coulomb for the gift of type 1 collagen. C. C.-M. was a recipient of a fellowships from the Fondation de France. This work was supported by grants from the Juvenile Diabetes Foundation International, the Association pour la Recherche sur le Cancer (ARC), and the Association Francaise des Diabe´ tiques.
2.3. In situ hybridization and immunohistochemistry For in situ hybridization, pancreatic rudiments were fixed in formalin 3.7% for 1 h, embedded in paraffin and sectioned. After removal of the paraffin, sections were treated with HCl 0.04 N for 30 min, washed at 658C in 2 £ sodium salt sodium citrate (SSC) for 20 min and permeabilized. Hybridization was carried out overnight at 478C in 50% formamide, 2 £ SSC pH 5, 5 £ Denhardt’s solution, 250 mg/ml yeast RNA, 500 mg/ml Herring sperm DNA) containing probe (1 mg/ml). Thereafter, the slides were washed with 2 £ SSC/50% formamide at 478C and then with decreasing concentrations of SSC buffer. Revelation was performed as described (Basmaciogullari et al., 2000). The cDNA used as template for riboprobe was the 141 bp PCR product corresponding to the rat FGFR1-IIIb specific exon amplified using primers described in Table 1. Immunohistochemistry was performed as previously described (Cras-Me´ neur et al., 2001). Acknowledgements We thank A. Basmaciogullari for advices with in situ
References Basmaciogullari, A., Cras-Me´ neur, C., Czernichow, P., Scharfmann, R., 2000. Pancreatic pattern of expression of thyrotropin-releasing hormone during rat embryonic development. J. Endocrinol. 166, 481– 488. Cras-Me´ neur, C., Elghazi, L., Czernichow, P., Scharfmann, R., 2001. Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differentiation. Diabetes 50, 1571–1579. Elghazi, L., Cras-Me´ neur, C., Czernichow, P., Scharfmann, R., 2002. Role for FGFR2IIIb-mediated signals in controlling pancreatic endocrine progenitor cell proliferation. Proc. Natl Acad. Sci. USA 99, 3884–3889. Pang, K., Mukonoweshuro, C., Wong, G., 1994. Beta cells arise from glucose transporter type 2 (Glut2)-expressing epithelial cells of the developing pancreas. Proc. Natl Acad. Sci. USA 91, 9559–9563. Teitelman, G., Alpert, S., Polak, J.M., Martinez, A., Hanahan, D., 1993. Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide. Development 118, 1031–1039. Zulewski, H., Abraham, E.J., Gerlach, M.J., Daniel, P.B., Moritz, W., Muller, B., Vallejo, M., Thomas, M.K., Habener, J.F., 2001. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 50, 521–533.
FGFR1-IIIb is a putative marker of pancreatic progenitor cells Corentin Cras-Me´neur, Raphael Scharfmann* INSERM U457, Hospital R. Debre´, 48, Boulevard Se´rurier, 75019 Paris, France Received 22 February 2002; received in revised form 18 April 2002; accepted 18 April 2002
Abstract The pancreas develops from buds that derive from the endodermal epithelium of the digestive tract. The progenitor cells that will give rise to the mature pancreatic cells reside within this epithelium. However, their exact identity remains unknown. In the present study, we searched for genes expressed by pancreatic progenitor cells. We focused our search on receptor tyrosine kinases. We found that fibroblast growth factor-IIIb (FGFR1-IIIb) expression is high in pancreatic epithelium enriched in progenitor cells. We next investigated FGFR1-IIIb expression throughout pancreatic development. At early stages of pancreas development, FGFR1-IIIb is expressed by pancreatic epithelial cells that resemble undifferentiated cells, while at later stages of development, FGFR1-IIIb expression decreases, concomitant with the expected decrease in the number of progenitor cells. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pancreas; Development; Fibroblast growth factor receptor; Progenitor cells
1. Results and discussion During the past years, different potential markers of pancreatic progenitor cells have been described such as tyrosine hydroxylase (Teitelman et al., 1993), the type 2 glucose transporter (Pang et al., 1994) or nestin (Zulewski et al., 2001). Up to now, none of these markers has been fully validated. We previously showed that when E13 rat pancreatic epithelium is kept in culture for 7 days with 1% fetal calf serum (FCS), the vast majority of the cells differentiate either into endocrine or acinar cells. When the epithelium is grown for 7 days in the presence of fibroblast growth factor (FGF-7) or endothelium growth factor (EGF), endocrine differentiation is repressed, and a large number of cells stain negative for endocrine and acinar markers. Based on retrospective analysis, these cells represent progenitor for endocrine cells (Cras-Me´neur et al., 2001; Elghazi et al., 2002). We selected candidate genes whose expression could be increased in epithelia rich in endocrine progenitor cells (uncultured E13.5 epithelium and E13.5 epithelium grown with FGF-7), when compared to epithelia depleted in progenitor cells (E13.5 epithelium grown without FGF-7). We first performed comparative polymerase chain reaction (PCR) amplification on cDNAs reverse transcribed either from E13.5 pancreatic epithelium or mesenchyme to define * Corresponding author. Tel.: 133-1400-31988; fax: 133-1404-09195. E-mail address: [email protected] (R. Scharfmann).
whether the candidate genes were expressed in the epithelial fraction. As shown in Fig. 1 ( left panel), some of the screened factors were amplified exclusively from the epithelial fraction (FGFR2-IIIb, FGFR4, FGFR1-IIIb and ErbB2). Other receptors were amplified in both the epithelial and mesenchymal fractions (FGFR1-IIIc, ErbB-1 and LIFR). We next compared the level of expression of these receptors in epithelia rich in endocrine progenitor cells (uncultured E13.5 epithelium and E13.5 epithelium grown with FGF-7) to epithelia depleted in progenitor cells (E13.5 epithelium grown without FGF-7). As shown in Fig. 1 (middle panel), the level of expression of FGFR1-IIIc, FGFR2-IIIb or LIFR did not show major variations in the different conditions analyzed. The expression of ErbB-2 decreased after 7 days in culture in either condition, while the expression of FGFR-4 was decreased in epithelia grown in the presence of FGF-7. The pattern of expression of FGFR1-IIIb was the most interesting. FGFR1-IIIb expression was enriched in uncultured epithelia or epithelia grown in the presence of FGF-7, when compared to epithelia cultured without FGF-7 (Fig. 1, middle panel). We next asked whether the increase in FGFR1-IIIb expression level also occurred in conditions where the pool of endocrine progenitor cells was expanded using EGF, known to amplify pancreatic progenitor cells (Cras-Me´neur et al., 2001). As shown in Fig. 1 (right panel), the level of expression of FGFR1-IIIb was increased in EGF-treated epithelia when compared to epithelia cultured in the absence of growth factor.
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00138-7
206
C. Cras-Me´ neur, R. Scharfmann / Mechanisms of Development 116 (2002) 205–208
Fig. 1. RT-PCR screening for candidate factors differentially expressed in epithelia enriched or poor in progenitor cells. Left panel: expression profiles of the different candidate factors in E13.5 epithelium and mesenchyme. Middle panel: expression profiles of the different candidate factors in uncultured E13.5 epithelium, epithelium cultured 7 days in control conditions or with FGF-7. Right panel: expression profile of FGFR1-IIIb in epithelia cultivated without or with EGF.
Fig. 2. Localization of the FGFR1-IIIb transcripts on sections of epithelia cultured for 7 days with FGF-7. (A) In situ hybridization was performed with a FGFR1-IIIb sense probe. (B) In situ hybridization was performed with a FGFR1-IIIb antisense probe. (C) Immunohistochemistry for insulin 1 glucagon (red) and amylase (green) on the section shown in B. (D) Reconstructed image composed of the images in shown in B and C. FGFR1-IIIb signal is shown in blue.
C. Cras-Me´ neur, R. Scharfmann / Mechanisms of Development 116 (2002) 205–208
To identify the cell types expressing FGFR1-IIIb, sections of epithelia grown with FGF-7 were hybridized for FGFR1-IIIb and then stained using anti-carboxypeptidase-A, anti-insulin and -glucagon antibodies. As shown in Fig. 2, the cells expressing FGFR1-IIIb transcripts, stain negative for acinar and endocrine markers. The same experiment was next performed on pancreatic
207
sections at different stages of development. FGFR1-IIIb transcripts were detected at E13.5, E16.5 and E18.5 but disappeared at later stages. At all stages analyzed, FGFR1-IIIb transcripts were detected only in epithelial structures. At E13.5, the vast majority of the FGFR1-IIIbexpressing cells stain negative for endocrine and acinar markers. Few cells coexpress FGFR1-IIIb and carboxypeptidase. At E16.5 and E18.5, few FGFR1-IIIb-expressing cells were detected that stain negative for endocrine and acinar markers. Later in development no FGFR1-IIIb expressing cells were detected in the pancreas (Fig. 3). In conclusion, we found that FGFR1-IIIb transcripts were enriched in epithelia grown in the presence of FGF-7 or EGF, when compared to epithelia grown in the absence of FGF-7 (or EGF), suggesting that FGFR1-IIIb represents a marker for progenitor cells. 2. Experimental procedures 2.1. Dissection and organ culture Dissections and cultures of pancreatic epithelia were performed as described previously (Cras-Me´ neur et al., 2001; Elghazi et al., 2002). 2.2. RNA extraction and comparative PCR Total RNA was extracted from pools of 30–40 pancreatic epithelia and reverse transcribed. To ensure semi-comparaTable 1 Oligonucleotides used for amplification Primer name 0
Fig. 3. Localization of the FGFR1-IIIb transcripts at different stages of the pancreatic development. Left panel: in situ hybridizations using a FGFR1IIIb antisense probe were performed on pancreatic sections from E13.5, E16.5, 18.5, 20, newborn and adult rats. Right panel: the sections first hybridized using a FGFR1-IIIb antisense probe, were next stained using anti-insulin 1 anti-glucagon antibodies (revealed in red) and anti-amylase antibody (revealed in green). Images were reconstructed and FGFR1-IIIb signal is presented in blue. Long arrows indicate cells that stain positive for FGFR1-IIIb and negative for endocrine and acinar markers. Arrowheads indicate cells that express both FGFR1-IIIb and amylase.
Insulin 5 Insulin 3 0 Glucagon-5 0 Glucagon-3 0 Amylase-5 0 Amylase-3 0 Cyclophilin-5 0 Cyclophilin-3 0 GAPDH-5 0 GAPDH-3 0 HNF-3b-5 0 HNF-3b-3 0 FGFR-1IIIc-5 0 FGFR-1IIIc-3 0 FGFR-2IIIb-5 0 FGFR-2IIIb-3 0 ErbB-2-5 0 ErbB-2-3 0 ErbB-1-5 0 ErbB-1-3 0 LIFR-5 0 LIFR-3 0 FGFR-4-5 0 FGFR-4-3 0 FGFR-1IIIb-5 0 FGFR-1IIIb-3 0
Primer sequence 5 0 -CCTAAGTGACCAGCTACA-3 0 5 0 -GTAGTTCTCCAGTTGGTA-3 0 5 0 -CTCAAGACACGGAGGAGAAC-3 0 5 0 -TTCACCAGCCAAGCAATGAAT-3 0 5 0 -TCGAACCAAGGTGGCTGACTA-3 0 5 0 -GGGCTCTGTCAGTAGGCACAA-3 0 5 0 -CAGGTCCTGGCATCTTGTCC-3 0 5 0 -TTGCTGGTCTTGCCATTCCT-3 0 5 0 -GTGATGCTGGTGCTGAGTATG-3 0 5 0 -AGTTGTCATGGATGACCTTGG-3 0 5 0 -TTGCTCCCTACGCCAATATGA-3 0 5 0 -GGGCACCTTGAGAAAGCTGTT-3 0 5 0 -TGGGAGCATTAACCACACCTACC-3 0 5 0 -GCACCTCCATTTCCTTGTCG-3 0 5 0 -GAGCACCGTACTGGACCAACAC-3 0 5 0 -TGGTAGGTGTGGTTGATGGACC-3 0 5 0 -AACTTGGAGCTTACCTACGTGCC-3 0 5 0 -TATCGACAGGAGCCAGTTGGTTA-3 0 5 0 -AAACTTGGAAATCACCTATGTGCAA-3 0 5 0 -CTCAGAAAGACATCTTGGACGATGT-3 0 5 0 -TACTGCCCACCCATCATTGAG-3 0 5 0 -TCTGTGGACCTTGGGGAATCT-3 0 5 0 -AAGGATTTGGCAGACCTGATC-3 0 5 0 -TGCACTTCCGAGACTCCAGATAC-3 0 5 0 -CGGGGATTAATAGCTCGGATG-3 0 5 0 -GCACAGGTCTGGTGACAGTGA-3 0
208
C. Cras-Me´ neur, R. Scharfmann / Mechanisms of Development 116 (2002) 205–208
tive amplification within non-saturating conditions, amplifications were performed on series of two-fold dilutions of the cDNAs as described (Basmaciogullari et al., 2000). Negative controls (without reverse transcription) did not show any amplification (data not shown). The oligonucleotides used for amplification are reported in Table 1.
hybridization and B. Coulomb for the gift of type 1 collagen. C. C.-M. was a recipient of a fellowships from the Fondation de France. This work was supported by grants from the Juvenile Diabetes Foundation International, the Association pour la Recherche sur le Cancer (ARC), and the Association Francaise des Diabe´ tiques.
2.3. In situ hybridization and immunohistochemistry For in situ hybridization, pancreatic rudiments were fixed in formalin 3.7% for 1 h, embedded in paraffin and sectioned. After removal of the paraffin, sections were treated with HCl 0.04 N for 30 min, washed at 658C in 2 £ sodium salt sodium citrate (SSC) for 20 min and permeabilized. Hybridization was carried out overnight at 478C in 50% formamide, 2 £ SSC pH 5, 5 £ Denhardt’s solution, 250 mg/ml yeast RNA, 500 mg/ml Herring sperm DNA) containing probe (1 mg/ml). Thereafter, the slides were washed with 2 £ SSC/50% formamide at 478C and then with decreasing concentrations of SSC buffer. Revelation was performed as described (Basmaciogullari et al., 2000). The cDNA used as template for riboprobe was the 141 bp PCR product corresponding to the rat FGFR1-IIIb specific exon amplified using primers described in Table 1. Immunohistochemistry was performed as previously described (Cras-Me´ neur et al., 2001). Acknowledgements We thank A. Basmaciogullari for advices with in situ
References Basmaciogullari, A., Cras-Me´ neur, C., Czernichow, P., Scharfmann, R., 2000. Pancreatic pattern of expression of thyrotropin-releasing hormone during rat embryonic development. J. Endocrinol. 166, 481– 488. Cras-Me´ neur, C., Elghazi, L., Czernichow, P., Scharfmann, R., 2001. Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differentiation. Diabetes 50, 1571–1579. Elghazi, L., Cras-Me´ neur, C., Czernichow, P., Scharfmann, R., 2002. Role for FGFR2IIIb-mediated signals in controlling pancreatic endocrine progenitor cell proliferation. Proc. Natl Acad. Sci. USA 99, 3884–3889. Pang, K., Mukonoweshuro, C., Wong, G., 1994. Beta cells arise from glucose transporter type 2 (Glut2)-expressing epithelial cells of the developing pancreas. Proc. Natl Acad. Sci. USA 91, 9559–9563. Teitelman, G., Alpert, S., Polak, J.M., Martinez, A., Hanahan, D., 1993. Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide. Development 118, 1031–1039. Zulewski, H., Abraham, E.J., Gerlach, M.J., Daniel, P.B., Moritz, W., Muller, B., Vallejo, M., Thomas, M.K., Habener, J.F., 2001. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 50, 521–533.