FGF2 Signaling in Mouse Embryonic Fibroblasts Is Crucial for ... - MDC
FGF2 Signaling in Mouse Embryonic Fibroblasts Is Crucial for ... - MDC
FGF2 Signaling in Mouse Embryonic Fibroblasts Is Crucial for ... - MDC
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Relative expression level54321+ <strong>FGF2</strong> 3 d– <strong>FGF2</strong> 3 d+ <strong>FGF2</strong> 5 d– <strong>FGF2</strong> 5 dRelative expression level654321+ <strong>FGF2</strong> 3 d– <strong>FGF2</strong> 3 d+ <strong>FGF2</strong> 5 d– <strong>FGF2</strong> 5 da0mcpt8plfgrem1il6<strong>in</strong>hbbtncgzmdtgfbimmp3Genesserp<strong>in</strong>e 1fgf7tnsf11saa3cxcl5cxcl14ccl2b0plfGenesbmp4Fig. 4. Validation of the global gene expression analysis with realtimePCR. a <strong>FGF2</strong>-dependent gene regulation of the secreted moleculesobserved <strong>in</strong> the microarray analysis was validated by realtimePCR <strong>in</strong> the presence or absence of <strong>FGF2</strong> <strong>for</strong> 3 or 5 days (as<strong>in</strong>dicated). b As <strong>in</strong> a . Omission of <strong>FGF2</strong> <strong>in</strong> the MEF culture waslead<strong>in</strong>g to decreased expression of prolifer<strong>in</strong>, whereas expressionof bmp4 was <strong>in</strong>creased around 2-fold. RNA expression was analy z e d a s d e s c r i b e d i n f i g u re 1b. d = D ay s .Validation of the <strong>FGF2</strong>-Dependent Gene Regulation<strong>in</strong> MEFsTo validate the expression of <strong>FGF2</strong>-regulated genesidentified <strong>in</strong> the microarray study with PMEF-N, we per<strong>for</strong>medreal-time PCR analyses with CF1 MEFs that arerout<strong>in</strong>ely used to culture H9 hESCs ( fig. 4 a). The expressionlevels <strong>for</strong> granzyme E, which was identified <strong>in</strong> themicroarray analysis, were not analyzed, s<strong>in</strong>ce it is highlyhomologous to granzyme D. MEFs were grown <strong>for</strong> 3 and5 days <strong>in</strong> stem cell medium with or without <strong>FGF2</strong>. Mostof the identified genes were regulated by <strong>FGF2</strong> as predictedby the microarray analysis. The strongest downregulationafter withdrawal of <strong>FGF2</strong> was observed <strong>in</strong> thegene expression level of mast cell protease 8 (mcpt8) andprolifer<strong>in</strong>. The genes <strong>for</strong> <strong>in</strong>hib<strong>in</strong> B and fgf7 showed onlya modest downregulation <strong>in</strong> the absence of <strong>FGF2</strong> by 50and 30%, respectively. Five of the genes, that is, mcpt8,prolifer<strong>in</strong>, greml<strong>in</strong> 1, tenasc<strong>in</strong> and tnsf11, showed significantlyhigher expression levels (around 3-fold) after 5days <strong>in</strong> <strong>FGF2</strong> medium compared to 3 days. This suggeststhat regulation of these genes is further <strong>in</strong>duced when theMEFs adapt to the culture conditions <strong>in</strong> stem cell medium.However, 4 of the genes studied, that is, saa3 andgenes <strong>for</strong> chemok<strong>in</strong>e ligands cxcl5, cxcl14 and ccl2, werenot significantly downregulated <strong>in</strong> the absence of <strong>FGF2</strong>under these conditions.Among the 17 identified genes, those <strong>for</strong> the moleculesInhib<strong>in</strong> B (Activ<strong>in</strong> B), Greml<strong>in</strong> 1 and FGF7 (KGF) areof special <strong>in</strong>terest, s<strong>in</strong>ce they regulate pathways whichhave already been shown to be <strong>in</strong>volved to keep hESCs <strong>in</strong>the undifferentiated state.Inhib<strong>in</strong> B, as a homodimer, <strong>for</strong>ms the ligand Activ<strong>in</strong>B and activates the transcription factors Smad2/3. Wehave shown previously that Smad2/3 are <strong>in</strong>deed activated[Besser, 2004] and their activation is required to ma<strong>in</strong>ta<strong>in</strong>hESCs undifferentiated [Amit et al., 2004; Beattie et al.,2005; James et al., 2005]. Interest<strong>in</strong>gly, Beattie et al. [2005]described that addition of FGF7 <strong>in</strong> feeder-free conditions<strong>in</strong> the presence of Activ<strong>in</strong> A and nicot<strong>in</strong>amide had astrong proliferative effect on hESCs. It was argued thatwhile Activ<strong>in</strong> A ma<strong>in</strong>ta<strong>in</strong>ed the pluripotent state underthose conditions, FGF7 was required <strong>for</strong> the proliferationof the cells, while nicot<strong>in</strong>amide was required to blockapoptosis [Beattie et al., 2005].Greml<strong>in</strong> 1 is an <strong>in</strong>hibitor of BMP signal<strong>in</strong>g [Hsu et al.,1998] and we have shown that this pathway is <strong>in</strong>hibited<strong>in</strong> the undifferentiated state [Besser, 2004; James et al.,2005]. In l<strong>in</strong>e with these observations, it has been describedthat activation of BMP signal<strong>in</strong>g <strong>in</strong> the undifferentiatedstate leads to the differentiation of hESCs <strong>in</strong>totrophoblasts [Xu et al., 2002]. Suppression of BMP signal<strong>in</strong>gis required to ma<strong>in</strong>ta<strong>in</strong> the pluripotent state [Xu et al.,2005]. This appears to be especially important whenhESCs are ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong> a pluripotent state with MEF-CM supplemented with KSR, s<strong>in</strong>ce this supplement conta<strong>in</strong>sBMP activity [Xu et al., 2005]. It has also been suggestedthat MEFs produce BMPs dur<strong>in</strong>g the condition<strong>in</strong>gphase and that the expression of bmp4 <strong>in</strong> the MEF cultureMa<strong>in</strong>tenance of Pluripotency by MEFsRequires <strong>FGF2</strong> <strong>Signal<strong>in</strong>g</strong>Cells Tissues Organs 2008;188:52–61 59
aESCM + <strong>FGF2</strong>ESCM – <strong>FGF2</strong>U0126DMSO1 2 3 4p-ERK1/2ERK1/2-Tubul<strong>in</strong>Relative expression levelb3.02.52.01.51.00.50+ <strong>FGF2</strong>– <strong>FGF2</strong>U0126DMSOmcpt8plfil6mmp3grem1Genes<strong>in</strong>hBgzmdtgficxcl14Fig. 5. Influence of MEK <strong>in</strong>hibitor on the expression of the <strong>FGF2</strong>-regulated genes <strong>in</strong> MEFs. a Western blot analysis of MEFs grownwith <strong>FGF2</strong> (+ <strong>FGF2</strong>) and treated with a specific MEK <strong>in</strong>hibitor(U0126), DMSO or cells grown without <strong>FGF2</strong> (–<strong>FGF2</strong>). Twentymicrograms of total prote<strong>in</strong> from whole cell extracts were probedw it h a nt i b o d ie s a g a i n s t pho s pho -E R K1/2 , E R K a nd -tubul<strong>in</strong> (asload<strong>in</strong>g control). Phosphorylation of ERK1/2 was decreased <strong>in</strong> theabsence of <strong>FGF2</strong>. U0126 was able to block phosphorylation ofERK1/2 almost completely, whereas the solvent control withDMSO showed no effect. b Real-time PCR analysis of MEFs culturedunder the <strong>in</strong>dicated conditions showed a downregulation ofthe target genes dependent on <strong>FGF2</strong> treatment. U0126 treatment<strong>in</strong> the presence of <strong>FGF2</strong> showed only an effect on some of the<strong>FGF2</strong> target genes, that is, mcpt8, plf, il6, mmp3, grem1 andtgf i. DMSO solvent control had no effect.is downregulated by <strong>FGF2</strong> [Greber et al., 2007]. To testthis possibility, we determ<strong>in</strong>ed the mRNA level <strong>for</strong> bmp4<strong>in</strong> the treated MEFs ( fig. 4 b). Indeed, while the mRNAlevels <strong>for</strong> prolifer<strong>in</strong> decreased <strong>in</strong> the absence of <strong>FGF2</strong>, theexpression level <strong>for</strong> bmp4 was <strong>in</strong>creased around 2-foldafter 3 days <strong>in</strong> culture without <strong>FGF2</strong>. After 5 days cultur<strong>in</strong>gwith <strong>FGF2</strong>, we observed a further decrease <strong>in</strong> bmp4expression as opposed to some of the <strong>FGF2</strong>-<strong>in</strong>duced geneswhere an <strong>in</strong>creased expression was observed.Effect of the MEK Inhibitor U0126 on <strong>FGF2</strong>-RegulatedGenesTo <strong>in</strong>vestigate how <strong>FGF2</strong> signal<strong>in</strong>g is promot<strong>in</strong>g the expressionof the target genes, we analyzed the activationstate of Akt and ERK signal<strong>in</strong>g [Eswarakumar et al., 2005].No difference <strong>in</strong> the phosphorylation of Akt <strong>in</strong> MEFstreated with or without <strong>FGF2</strong> was observed <strong>in</strong> Westernblot analysis us<strong>in</strong>g a phosphospecific Akt antibody, suggest<strong>in</strong>gthat <strong>FGF2</strong> does not regulate Akt activity underthese conditions (data not shown). The phosphorylation ofERK1/2, on the other hand, changed depend<strong>in</strong>g on <strong>FGF2</strong>treatment ( fig. 5 a). We there<strong>for</strong>e used the specific <strong>in</strong>hibitorof MEK1/2 signal<strong>in</strong>g (U0126) [Favata et al., 1998] to <strong>in</strong>hibitthe activation of ERKs. The <strong>in</strong>hibitor U0126 was ableto block the phosphorylation of ERK1/2 almost completely,even stronger than observed by withdrawal of <strong>FGF2</strong>. Asa control, treatment with DMSO showed no effects of thephosphorylation pattern ( fig. 5 a). Based on these results,we analyzed the expression of the <strong>FGF2</strong>-regulated genesby real-time PCR. As shown be<strong>for</strong>e <strong>in</strong> figure 3 a, the level<strong>for</strong> mcpt8, prolifer<strong>in</strong>, <strong>in</strong>hib<strong>in</strong> B, greml<strong>in</strong> 1 and fgf7 decreased<strong>in</strong> the absence of <strong>FGF2</strong>. Note that this experimentwas carried out over 3 days to avoid toxic effects of the <strong>in</strong>hibitor( fig. 5 b). Interest<strong>in</strong>gly, U0126 treatment showed aneffect on some of the <strong>FGF2</strong> target genes (mcpt8, plf, il6,mmp3, grem1 and tgf i) but not on others (<strong>in</strong>h B andgzmd). In addition, decreased gene expression by <strong>FGF2</strong>withdrawal was <strong>in</strong> all cases more pronounced than the decrease<strong>in</strong> the presence of U0126. The drop <strong>in</strong> phospho-ERK levels was stronger <strong>in</strong> the presence of U0126 than bywithdrawal of <strong>FGF2</strong>. Thus, these results suggest that <strong>FGF2</strong>signal<strong>in</strong>g acts <strong>in</strong> part <strong>in</strong>dependently of the Ras/ERK pathway<strong>in</strong> MEFs ( fig. 5 b). Further experiments are necessaryto determ<strong>in</strong>e how <strong>FGF2</strong> elicits the regulation of targetgenes <strong>in</strong> MEFs under these conditions.S u m m a r yThis study shows that <strong>FGF2</strong> signal<strong>in</strong>g is required <strong>for</strong>MEFs to produce secreted factors support<strong>in</strong>g the undifferentiatedgrowth of hESCs. No effect of the modulation60Cells Tissues Organs 2008;188:52–61Diecke/Quiroga-Negreira/Redmer/Besser
of <strong>FGF2</strong> signal<strong>in</strong>g directly <strong>in</strong> the hESCs was observed.Based on these observations, we identified 17 genes regulatedby <strong>FGF2</strong> by a microarray analysis, of which 13 wereconfirmed by real-time PCR <strong>in</strong> MEFs from the CF1mouse stra<strong>in</strong>. Among these genes we identified factors,that is Inhib<strong>in</strong> B, Greml<strong>in</strong> 1 and FGF7, that have beenpreviously implicated <strong>in</strong> the ma<strong>in</strong>tenance of hESC pluripotency.The results presented <strong>in</strong> this study will be important<strong>for</strong> the development of culture protocols <strong>for</strong>hESCs replac<strong>in</strong>g the undef<strong>in</strong>ed CM from MEFs by mediacompositions with controlled recomb<strong>in</strong>ant factors free ofcontam<strong>in</strong>at<strong>in</strong>g animal material.AcknowledgementsWe would like to thank Ali H. Brivanlou <strong>for</strong> his support dur<strong>in</strong>gthe microarray analysis <strong>in</strong> his laboratory at Rockefeller Universityand Agnés Viale at the genomic core facility of MSKCC <strong>for</strong>her support <strong>in</strong> this analysis. We also thank Françoise André <strong>for</strong>excellent technical support and Johannes Fritzmann, ManfredGossen and Inés Ibañez Tallon <strong>for</strong> discussion and critical read<strong>in</strong>gof the manuscript. We would like to thank the WiCell ResearchInstitute <strong>for</strong> provid<strong>in</strong>g the H9 cell l<strong>in</strong>e. This study was supportedby an EC Marie Curie IRG (FP6-021407) and a DFG grant (BE3257/1-1; to D.B.).ReferencesAmit, M., M.K. Carpenter, M.S. Inokuma, C.P.Chiu, C.P. Harris, M.A. Waknitz, J. Itskovitz-Eldor,J.A. Thomson (2000) Clonally derivedhuman embryonic stem cell l<strong>in</strong>esma<strong>in</strong>ta<strong>in</strong> pluripotency and proliferative potential<strong>for</strong> prolonged periods of culture. DevBiol 227: 271–278.Amit, M., C. Shariki, V. Margulets, J. Itskovitz-Eldor (2004) Feeder layer- and serum-freeculture of human embryonic stem cells. BiolReprod 70: 837–845.Arman, E., R. Haffner-Krausz, Y. Chen, J.K.Heath, P. Lonai (1998) Targeted disruption offibroblast growth factor (FGF) receptor 2suggests a role <strong>for</strong> FGF signal<strong>in</strong>g <strong>in</strong> pregastrulationmammalian development. ProcNatl Acad Sci USA 95: 5082–5087.Beattie, G.M., A.D. Lopez, N. Bucay, A. H<strong>in</strong>ton,M.T. Firpo, C.C. K<strong>in</strong>g, A. Hayek (2005) Activ<strong>in</strong>A ma<strong>in</strong>ta<strong>in</strong>s pluripotency of humanembryonic stem cells <strong>in</strong> the absence of feederlayers. Stem Cells 23: 489–495.Besser, D. (2004) Expression of nodal, lefty-A,and lefty-B <strong>in</strong> undifferentiated human embryonicstem cells requires activation ofSmad2/3. J Biol Chem 279: 45076–45084.Chambers, I., D. Colby, M. Robertson, J. Nichols,S. Lee, S. Tweedie, A. Smith (2003) Functionalexpression clon<strong>in</strong>g of Nanog, a pluripotencysusta<strong>in</strong><strong>in</strong>g factor <strong>in</strong> embryonic stemcells. Cell 113: 643–655.Eswarakumar, V.P., I. Lax, J. Schless<strong>in</strong>ger (2005)Cellular signal<strong>in</strong>g by fibroblast growth factorreceptors. Cytok<strong>in</strong>e Growth Factor Rev16: 139–149.Favata, M.F., K.Y. Horiuchi, E.J. Manos, A.J.Daulerio, D.A. Stradley, W.S. Feeser, D.E.Van Dyk, W.J. Pitts, R.A. Earl, F. Hobbs, R.A.Copeland, R.L. Magolda, P.A. Scherle, J.M.Trzaskos (1998) Identification of a novel <strong>in</strong>hibitorof mitogen-activated prote<strong>in</strong> k<strong>in</strong>asek<strong>in</strong>ase. J Biol Chem 273: 18623–18632.Greber, B., H. Lehrach, J. Adjaye (2007) Fibroblastgrowth factor 2 modulates trans<strong>for</strong>m<strong>in</strong>ggrowth factor signal<strong>in</strong>g <strong>in</strong> mouseembryonic fibroblasts and human ESCs(hESCs) to support hESC self-renewal. StemCells 25: 455–464.Hay, D.C., L. Sutherland, J. Clark, T. Burdon(2004) Oct-4 knockdown <strong>in</strong>duces similarpatterns of endoderm and trophoblast differentiationmarkers <strong>in</strong> human and mouseembryonic stem cells. Stem Cells 22: 225–235.Hollnagel, A., V. Oehlmann, J. Heymer, U.Ruther, A. Nordheim (1999) Id genes are directtargets of bone morphogenetic prote<strong>in</strong><strong>in</strong>duction <strong>in</strong> embryonic stem cells. J BiolChem 274: 19838–19845.Hsu, D.R., A.N. Economides, X. Wang, P.M. Eimon,R.M. Harland (1998) The Xenopus dorsaliz<strong>in</strong>gfactor Greml<strong>in</strong> identifies a novelfamily of secreted prote<strong>in</strong>s that antagonizeBMP activities. Mol Cell 1: 673–683.Humphrey, R.K., G.M. Beattie, A.D. Lopez, N.Bucay, C.C. K<strong>in</strong>g, M.T. Firpo, S. Rose-John,A. Hayek (2004) Ma<strong>in</strong>tenance of pluripotency<strong>in</strong> human embryonic stem cells is STAT3<strong>in</strong>dependent. Stem Cells 22: 522–530.James, D., A.J. Lev<strong>in</strong>e, D. Besser, A. Hemmati-Brivanlou (2005) TGF /Activ<strong>in</strong>/Nodal signal<strong>in</strong>gis necessary <strong>for</strong> the ma<strong>in</strong>tenance ofpluripotency <strong>in</strong> human embryonic stemcells. Development 132: 1273–1282.Levenste<strong>in</strong>, M.E., T.E. Ludwig, R.H. Xu, R.A.Llanas, K. VanDenHeuvel-Kramer, D. Mann<strong>in</strong>g,J.A. Thomson (2006) Basic fibroblastgrowth factor support of human embryonicstem cell self-renewal. Stem Cells 24: 568–574.Mitsui, K., Y. Tokuzawa, H. Itoh, K. Segawa, M.Murakami, K. Takahashi, M. Maruyama, M.Maeda, S. Yamanaka (2003) The homeoprote<strong>in</strong>Nanog is required <strong>for</strong> ma<strong>in</strong>tenance ofpluripotency <strong>in</strong> mouse epiblast and ES cells.Cell 113: 631–642.Nichols, J., B. Zevnik, K. Anastassiadis, H. Niwa,D. Klewe-Nebenius, I. Chambers, H. Scholer,A. Smith (1998) Formation of pluripotentstem cells <strong>in</strong> the mammalian embryo dependson the POU transcription factor Oct4.Cell 95: 379–391.Puceat, M. (2007) TGF <strong>in</strong> the differentiation ofembryonic stem cells. Cardiovasc Res 74:256–261.Reub<strong>in</strong>off, B.E., M.F. Pera, C.Y. Fong, A. Trounson,A. Bongso (2000) <strong>Embryonic</strong> stem celll<strong>in</strong>es from human blastocysts: somatic differentiation<strong>in</strong> vitro. Nat Biotechnol 18: 399–404.Sato, N., L. Meijer, L. Skaltsounis, P. Greengard,A.H. Brivanlou (2004) Ma<strong>in</strong>tenance of pluripotency<strong>in</strong> human and mouse embryonicstem cells through activation of Wnt signal<strong>in</strong>gby a pharmacological GSK-3-specific <strong>in</strong>hibitor.Nat Med 10: 55–63.Smith, A.G. (2001) Embryo-derived stem cells:of mice and men. Annu Rev Cell Dev Biol 17:435–462.Tanaka S., T. Kunath, A.K. Hadjantonakis, A.Nagy, J. Rossant (1998) Promotion of trophoblaststem cell proliferation by FGF4. Science282: 2072–2075.Vallier, L., M. Alexander, R.A. Pedersen (2005)Activ<strong>in</strong>/Nodal and FGF pathways cooperateto ma<strong>in</strong>ta<strong>in</strong> pluripotency of human embryonicstem cells. J Cell Sci 118: 4495–4509.Xu, C., M.S. Inokuma, J. Denham, K. Golds, P.Kundu, J.D. Gold, M.K. Carpenter (2001)Feeder-free growth of undifferentiated humanembryonic stem cells. Nat Biotechnol19: 971–974.Xu, R.H., X. Chen, D.S. Li, R. Li, G.C. Addicks,C. Glennon, T.P. Zwaka, J.A. Thomson(2002) BMP4 <strong>in</strong>itiates human embryonicstem cell differentiation to trophoblast. NatBiotechnol 20: 1261–1264.Xu, R.H., R.M. Peck, D.S. Li, X. Feng, T. Ludwig,J.A. Thomson (2005) Basic FGF and suppressionof BMP signal<strong>in</strong>g susta<strong>in</strong> undifferentiatedproliferation of human ES cells. NatMethods 2: 18 5 –19 0 .Ma<strong>in</strong>tenance of Pluripotency by MEFsRequires <strong>FGF2</strong> <strong>Signal<strong>in</strong>g</strong>Cells Tissues Organs 2008;188:52–61 61
S. Diecke et al. Ma<strong>in</strong>tenance of Pluripotency by MEFs requires <strong>FGF2</strong> signal<strong>in</strong>gSupplementary MaterialSequences of upstream and downstream primers <strong>for</strong> genes analyzed by realtimeRT-PCR (h – human, m – mouse) are listed below <strong>in</strong>clud<strong>in</strong>g the length ofthe expected product <strong>in</strong> bases pairs (bp; Length).Gene Forward Primer Reverse Primer Lengthlefty-A (h) 5’-gggaattgggatacctggat-3’ 5’-ctaaatatgcacgggcaagg-3’ 206 bpoct4 (h) 5’-gaaggatgtggtccgagtgt-3’ 5’-gtgacagagacagggggaaa-3’ 243 bpnanog (h) 5’-accagaactgtgttctcttccacc-3’ 5’-ggttgctccaggttgaattgttcc-3’ 333 bp-act<strong>in</strong> (h) 5’-tggcaccacaccttctacaatgagc-3’ 5’-gcacagcttctccttaatgtcacgc-3’ 400 bpnodal (h) 5’-agacatcatccgcagcctaca-3’ 5’-gacctgggacaaagtgacagtgaa -3’ 268 bpmsx2 (h) 5’-aaatctggttccagaaccgaaggg-3’ 5’-atgggaagcacaggtctatggaacg-3’ 187 bp<strong>in</strong>hbb (m) 5’-tcttcatcgactttcggctcatcg-3’ 5’-caaagtagagcatggacatggagc-3’ 234 bpmcpt8 (m) 5’-atgacatcatgctcctgaagctgg-3’ 5’-caaagtagagcatggacatggagc-3’ 225 bpgzmd (m) 5’-aacgtttccgacactacactgagacc-3’ 5’-ttcatgttcctgcttatccacggc-3’ 208 bpsaa3 (m) 5’-atcttgatcctgggagttgacagc-3’ 5’-atgtcccgtgaacttctgaacagc-3’ 237 bpil6 (m) 5’-tctggtcttctggagtaccatagc-3’ 5’-attggatggtcttggtccttagcc-3’ 232 bpplf (m) 5’-tgttgccttatttcccatgtgtgc-3’ 5’-aagagctgaatagtgtgtgagcctgg-3’ 250 bptnfsf11 (m) 5’-atcgctctgttcctgtactttcgagc-3’ 5’-atgtgttcgagttccttctgcacg-3’ 206 bpcxcl14 (m) 5’-tccaagtgtaagtgttccaggaaggg-3’ 5’-ttgatgaagcgtttggtgctctgc-3’ 188 bpmmp3 (m) 5’-aagggtggatgctgtctttgaagc-3’ 5’-gccatagcacatgctgaacaaagc-3’ 230 bpgrem1 (m) 5’-taagtcaaagcgggcacattcagc-3’ 5’-tgccttctagcaaaggctgtttcc-3’ 214 bptnc (m) 5’-aagactgcaaagagcaaaggtgcc-3’ 5’-acacacattgtccttggttgctgc-3’ 150 bpcxcl5 (m) 5’-tggcatttctgttgctgttcacgc-3’ 5’-tgcagggatcacctccaaattagc-3’ 152 bpfgf7 (m) 5’-aatgacatgagtccggagcaaacg-3’ 5’-attgccacaattccaactgccacg-3’ 232 bpserp<strong>in</strong>e1 (m) 5’-tgcctgacatgtttagtgcaaccc-3’ 5’-tgtgccgaaccacaaagagaaagg-3’ 210 bptgfbi (m) 5’-tgagcaacgtcaacatcgaactgc-3’ 5’-accttgtcaatgagatgcaccacg-3’ 226 bpccl2 (m) 5’-ttgtcaccaagctcaagagagagg-3’ 5’-tacagaagtgctgaggtggtgtgg-3’ 224 bpbmp4 (m) 5’-agaagaataagaactgccgtcgcc-3’ 5’-atggcatggttggttgagttgagg-3’ 160 bp-24-