The successive events that cells experience throughout development shape their intrinsic capacity to respond and integrate RTK inputs. Cellular responses to RTKs rely on different mechanisms of regulation that establish proper levels of RTK activation, define duration of RTK action, and exert quantitative/qualitative signalling outcomes. The extent to which cells are competent to deal with fluctuations in RTK signalling is incompletely understood. Here, we employ a genetic system to enhance RTK signalling in a tissue-specific manner. The chosen RTK is the hepatocyte growth factor (HGF) receptor Met, an appropriate model due to its pleiotropic requirement in distinct developmental events. Ubiquitously enhanced Met in Cre/loxP-based Rosa26stopMet knock-in context (Del-R26Met) reveals that most tissues are capable of buffering enhanced Met-RTK signalling thus avoiding perturbation of developmental programs. Nevertheless, this ubiquitous increase of Met does compromise selected programs such as myoblast migration. Using cell-type specific Cre drivers, we genetically showed that altered myoblast migration results from ectopic Met expression in limb mesenchyme rather than in migrating myoblasts themselves. qRT-PCR analyses show that ectopic Met in limbs causes molecular changes such as downregulation in the expression levels of Notum and Syndecan4, two known regulators of morphogen gradients. Molecular and functional studies revealed that ectopic Met expression in limb mesenchyme does not alter HGF expression patterns and levels, but impairs HGF bioavailability. Together, our findings show that myoblasts, in which Met is endogenously expressed, are capable of buffering increased RTK levels, and identify mesenchymal cells as a cell type vulnerable to ectopic Met-RTK signalling. These results illustrate that embryonic cells are sensitive to alterations in the spatial distribution of RTK action, yet resilient to fluctuations in signalling levels of an RTK when occurring in its endogenous domain of activity.
References
[1]
Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2010;141(7):1117–34. doi: 10.1016/j.cell.2010.06.011. pmid:20602996
[2]
Casaletto JB, McClatchey AI. Spatial regulation of receptor tyrosine kinases in development and cancer. Nat Rev Cancer. 2012;12(6):387–400. doi: 10.1038/nrc3277. pmid:22622641
[3]
Bache KG, Slagsvold T, Stenmark H. Defective downregulation of receptor tyrosine kinases in cancer. Embo J. 2004;23(14):2707–12. pmid:15229652 doi: 10.1038/sj.emboj.7600292
[4]
Maina F. Strategies to overcome drug resistance of receptor tyrosine kinaseaddicted cancer cells. Current medicinal chemistry. 2014;21(14):1607–17. pmid:23992334 doi: 10.2174/09298673113209990222
[5]
Flores GV, Duan H, Yan H, Nagaraj R, Fu W, Zou Y, et al. Combinatorial signaling in the specification of unique cell fates. Cell. 2000;103(1):75–85. pmid:11051549 doi: 10.1016/s0092-8674(00)00106-9
[6]
de Celis JF, Bray S, Garcia-Bellido A. Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing. Development. 1997;124(10):1919–28. pmid:9169839
[7]
Li J, Li WX. Drosophila gain-of-function mutant RTK torso triggers ectopic Dpp and STAT signaling. Genetics. 2003;164(1):247–58. pmid:12750336
[8]
Trusolino L, Bertotti A, Comoglio PM. MET signalling: principles and functions in development, organ regeneration and cancer. Nat Rev Mol Cell Biol. 2010;11(12):834–48. doi: 10.1038/nrm3012. pmid:21102609
[9]
Huh CG, Factor VM, Sanchez A, Uchida K, Conner EA, Thorgeirsson SS. Hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair. Proc Natl Acad Sci U S A. 2004;101(13):4477–82. pmid:15070743. doi: 10.1073/pnas.0306068101
[10]
Calvi C, Podowski M, Lopez-Mercado A, Metzger S, Misono K, Malinina A, et al. Hepatocyte growth factor, a determinant of airspace homeostasis in the murine lung. PLoS genetics. 2013;9(2):e1003228. doi: 10.1371/journal.pgen.1003228. pmid:23459311;
[11]
Chmielowiec J, Borowiak M, Morkel M, Stradal T, Munz B, Werner S, et al. c-Met is essential for wound healing in the skin. J Cell Biol. 2007;177(1):151–62. pmid:17403932 doi: 10.1083/jcb.200701086
[12]
Knudsen BS, Vande Woude G. Showering c-MET-dependent cancers with drugs. Curr Opin Genet Dev. 2008;18(1):87–96. doi: 10.1016/j.gde.2008.02.001. pmid:18406132
[13]
Gherardi E, Birchmeier W, Birchmeier C, Vande Woude G. Targeting MET in cancer: rationale and progress. Nat Rev Cancer. 2012;12(2):89–103. Epub 2012/01/25. doi: 10.1038/nrc3205. pmid:22270953
[14]
Straussman R, Morikawa T, Shee K, Barzily-Rokni M, Qian ZR, Du J, et al. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature. 2012;487(7408):500–4. doi: 10.1038/nature11183. pmid:22763439
[15]
Bladt F, Riethmacher D, Isenmann S, Aguzzi A, Birchmeier C. Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud. Nature. 1995;376:768–71. pmid:7651534 doi: 10.1038/376768a0
[16]
Schmidt C, Bladt F, Goedecke S, Brinkmann V, Zschlesche W, Sharpe M, et al. Scatter factor/hepatocyte growth factor is essential for liver development. Nature. 1995;373:699–702. pmid:7854452 doi: 10.1038/373699a0
[17]
Uehara Y, Minowa O, Mori C, Shiota K, Kuno J, Noda T, et al. Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor. Nature. 1995;373:702–5. pmid:7854453 doi: 10.1038/373702a0
[18]
Maina F, Casagranda F, Audero E, Simeone A, Comoglio P, Klein R, et al. Uncoupling of Grb2 from the Met receptor in vivo reveals complex roles in muscle development. Cell. 1996;87:531–42. pmid:8898205 doi: 10.1016/s0092-8674(00)81372-0
[19]
Maina F, Hilton MC, Andres R, Wyatt S, Klein R, Davies AM. Multiple roles for hepatocyte growth factor in sympathetic neuron development. Neuron. 1998;20:835–46. pmid:9620689 doi: 10.1016/s0896-6273(00)80466-3
[20]
Maina F, Hilton MC, Ponzetto C, Davies AM, Klein R. Met receptor signaling is required for sensory nerve development and HGF promotes axonal growth and survival of sensory neurons. Genes and Development. 1997;11:3341–50. pmid:9407027 doi: 10.1101/gad.11.24.3341
[21]
Maina F, Klein R. Hepatocyte growth factor—a versatile signal for developing neurons. Nature Neuroscience. 1999;2:213–7. pmid:10195212
[22]
Lamballe F, Genestine M, Caruso N, Arce V, Richelme S, Helmbacher F, et al. Pool-specific regulation of motor neuron survival by neurotrophic support. J Neurosci. 2011;31(31):11144–58. doi: 10.1523/JNEUROSCI.2198-11.2011. pmid:21813676
[23]
Caruso N, Herberth B, Lamballe F, Arce-Gorvel V, Maina F, Helmbacher F. Plasticity versus specificity in RTK signalling modalities for distinct biological outcomes in motor neurons. BMC Biol. 2014;12(1):56. doi: 10.1186/s12915-014-0056-6
[24]
Genestine M, Caricati E, Fico A, Richelme S, Hassani H, Sunyach C, et al. Enhanced neuronal Met signalling levels in ALS mice delay disease onset. Cell Death Dis. 2011;2:e130. doi: 10.1038/cddis.2011.11. pmid:21412276
[25]
Tonges L, Ostendorf T, Lamballe F, Genestine M, Dono R, Koch JC, et al. Hepatocyte growth factor protects retinal ganglion cells by increasing neuronal survival and axonal regeneration in vitro and in vivo. J Neurochem. 2011;117(5):892–903. pmid:21443522. doi: 10.1111/j.1471-4159.2011.07257.x.
[26]
Schwenk F, Baron U, Rajewsky K. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 1995;23:5080–1. pmid:8559668 doi: 10.1093/nar/23.24.5080
[27]
Kelly R, Alonso S, Tajbakhsh S, Cossu G, Buckingham M. Myosin light chain 3F regulatory sequences confer regionalized cardiac and skeletal muscle expression in transgenic mice. J Cell Biol. 1995;129(2):383–96. Epub 1995/04/01. pmid:7721942 doi: 10.1083/jcb.129.2.383
[28]
Maina F, Pante G, Helmbacher F, Andres R, Porthin A, Davies AM, et al. Coupling Met to specific pathways results in distinct developmental outcomes. Mol Cell. 2001;7(6):1293–306. pmid:11430831 doi: 10.1016/s1097-2765(01)00261-1
[29]
Buckingham M, Rigby PW. Gene regulatory networks and transcriptional mechanisms that control myogenesis. Dev Cell. 2014;28(3):225–38. doi: 10.1016/j.devcel.2013.12.020. pmid:24525185
[30]
Engleka KA, Gitler AD, Zhang M, Zhou DD, High FA, Epstein JA. Insertion of Cre into the Pax3 locus creates a new allele of Splotch and identifies unexpected Pax3 derivatives. Dev Biol. 2005;280(2):396–406. Epub 2005/05/11. pmid:15882581 doi: 10.1016/j.ydbio.2005.02.002
[31]
Logan M, Martin JF, Nagy A, Lobe C, Olson EN, Tabin CJ. Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis. 2002;33(2):77–80. Epub 2002/07/12. pmid:12112875 doi: 10.1002/gene.10092
[32]
Luxardi G, Galli A, Forlani S, Lawson K, Maina F, Dono R. Glypicans are differentially expressed during patterning and neurogenesis of early mouse brain. Biochem Biophys Res Commun. 2007;352(1):55–60. pmid:17107664 doi: 10.1016/j.bbrc.2006.10.185
[33]
Fico A, de Chevigny A, Egea J, Bosl MR, Cremer H, Maina F, et al. Modulating Glypican4 Suppresses Tumorigenicity of Embryonic Stem Cells while Preserving Self-Renewal and Pluripotency. Stem Cells. 2012. Epub 2012/07/05. doi: 10.1002/stem.1165
[34]
Fico A, de Chevigny A, Melon C, Bohic M, Kerkerian-Le Goff L, Maina F, et al. Reducing glypican-4 in ES cells improves recovery in a rat model of Parkinson's disease by increasing the production of dopaminergic neurons and decreasing teratoma formation. J Neurosci. 2014;34(24):8318–23. doi: 10.1523/JNEUROSCI.2501-13.2014. pmid:24920634
[35]
Tkachenko E, Rhodes JM, Simons M. Syndecans: new kids on the signaling block. Circ Res. 2005;96(5):488–500. pmid:15774861 doi: 10.1161/01.res.0000159708.71142.c8
[36]
Wang X, Page-McCaw A. A matrix metalloproteinase mediates long-distance attenuation of stem cell proliferation. J Cell Biol. 2014;206(7):923–36. doi: 10.1083/jcb.201403084. pmid:25267296
[37]
Hacker U, Nybakken K, Perrimon N. Heparan sulphate proteoglycans: the sweet side of development. Nat Rev Mol Cell Biol. 2005;6(7):530–41. pmid:16072037 doi: 10.1038/nrm1681
[38]
Kakugawa S, Langton PF, Zebisch M, Howell SA, Chang TH, Liu Y, et al. Notum deacylates Wnt proteins to suppress signalling activity. Nature. 2015;519(7542):187–92. doi: 10.1038/nature14259. pmid:25731175
[39]
Karihaloo A, Kale S, Rosenblum ND, Cantley LG. Hepatocyte growth factor-mediated renal epithelial branching morphogenesis is regulated by glypican-4 expression. Mol Cell Biol. 2004;24(19):8745–52. pmid:15367691 doi: 10.1128/mcb.24.19.8745-8752.2004
[40]
Cecchi F, Pajalunga D, Fowler CA, Uren A, Rabe DC, Peruzzi B, et al. Targeted disruption of heparan sulfate interaction with hepatocyte and vascular endothelial growth factors blocks normal and oncogenic signaling. Cancer Cell. 2012;22(2):250–62. doi: 10.1016/j.ccr.2012.06.029. pmid:22897854
[41]
Derksen PW, Keehnen RM, Evers LM, van Oers MH, Spaargaren M, Pals ST. Cell surface proteoglycan syndecan-1 mediates hepatocyte growth factor binding and promotes Met signaling in multiple myeloma. Blood. 2002;99(4):1405–10. pmid:11830493 doi: 10.1182/blood.v99.4.1405
[42]
Pante G, Thompson J, Lamballe F, Iwata T, Ferby I, Barr FA, et al. Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth. J Cell Biol. 2005;171(2):337–48. pmid:16247031 doi: 10.1083/jcb.200502013
[43]
Gerritsen ME, Tomlinson JE, Zlot C, Ziman M, Hwang S. Using gene expression profiling to identify the molecular basis of the synergistic actions of hepatocyte growth factor and vascular endothelial growth factor in human endothelial cells. Br J Pharmacol. 2003;140(4):595–610. Epub 2003/09/25. pmid:14504135 doi: 10.1038/sj.bjp.0705494
[44]
Factor VM, Seo D, Ishikawa T, Kaposi-Novak P, Marquardt JU, Andersen JB, et al. Loss of c-Met disrupts gene expression program required for G2/M progression during liver regeneration in mice. PLoS One. 2010;5(9). Epub 2010/09/24. doi: 10.1371/journal.pone.0012739
[45]
Vasyutina E, Stebler J, Brand-Saberi B, Schulz S, Raz E, Birchmeier C. CXCR4 and Gab1 cooperate to control the development of migrating muscle progenitor cells. Genes Dev. 2005;19(18):2187–98. pmid:16166380 doi: 10.1101/gad.346205
[46]
Moumen A, Ieraci A, Patane S, Sole C, Comella JX, Dono R, et al. Met signals hepatocyte survival by preventing Fas-triggered FLIP degradation in a PI3k-Akt-dependent manner. Hepatology. 2007;45(5):1210–7. pmid:17464994. doi: 10.1002/hep.21604
[47]
Moumen A, Patane S, Porras A, Dono R, Maina F. Met acts on Mdm2 via mTOR to signal cell survival during development. Development. 2007;134(7):1443–51. pmid:17329361 doi: 10.1242/dev.02820
[48]
Furlan A, Lamballe F, Stagni V, Hussain A, Richelme S, Prodosmo A, et al. Met acts through Abl to regulate p53 transcriptional outcomes and cell survival in the developing liver. J Hepatol. 2012;57(6):1292–8. Epub 2012/08/15. doi: 10.1016/j.jhep.2012.07.044. pmid:22889954
[49]
Patane S, Pietrancosta N, Hassani H, Leroux V, Maigret B, Kraus JL, et al. A new Met inhibitory-scaffold identified by a focused forward chemical biological screen. Biochem Biophys Res Commun. 2008;375(2):184–9. doi: 10.1016/j.bbrc.2008.07.159. pmid:18703015
[50]
Furlan A, Colombo F, Kover A, Issaly N, Tintori C, Angeli L, et al. Identification of new aminoacid amides containing the imidazo[2,1-b]benzothiazol-2-ylphenyl moiety as inhibitors of tumorigenesis by oncogenic Met signaling. Eur J Med Chem. 2012;47(1):239–54. Epub 2011/12/06. doi: 10.1016/j.ejmech.2011.10.051. pmid:22138308
[51]
Relaix F, Polimeni M, Rocancourt D, Ponzetto C, Schafer BW, Buckingham M. The transcriptional activator PAX3-FKHR rescues the defects of Pax3 mutant mice but induces a myogenic gain-of-function phenotype with ligand-independent activation of Met signaling in vivo. Genes Dev. 2003;17(23):2950–65. pmid:14665670 doi: 10.1101/gad.281203
[52]
Gutierrez J, Cabrera D, Brandan E. Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells. Skeletal muscle. 2014;4(1):5. doi: 10.1186/2044-5040-4-5. pmid:24517345
[53]
Cornelison DD, Filla MS, Stanley HM, Rapraeger AC, Olwin BB. Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration. Dev Biol. 2001;239(1):79–94. Epub 2002/01/11. pmid:11784020 doi: 10.1006/dbio.2001.0416
[54]
Lee SJ, Huynh TV, Lee YS, Sebald SM, Wilcox-Adelman SA, Iwamori N, et al. Role of satellite cells versus myofibers in muscle hypertrophy induced by inhibition of the myostatin/activin signaling pathway. Proc Natl Acad Sci U S A. 2012;109(35):E2353–60. Epub 2012/08/08. doi: 10.1073/pnas.1206410109. pmid:22869749
[55]
Rodgers JT, King KY, Brett JO, Cromie MJ, Charville GW, Maguire KK, et al. mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature. 2014;510(7505):393–6. doi: 10.1038/nature13255. pmid:24870234
[56]
Caruso N, Herberth B, Bartoli M, Puppo F, Dumonceaux J, Zimmermann A, et al. Deregulation of the protocadherin gene FAT1 alters muscle shapes: implications for the pathogenesis of facioscapulohumeral dystrophy. PLoS genetics. 2013;9(6):e1003550. doi: 10.1371/journal.pgen.1003550. pmid:23785297
[57]
Barber TD, Barber MC, Tomescu O, Barr FG, Ruben S, Friedman TB. Identification of target genes regulated by PAX3 and PAX3-FKHR in embryogenesis and alveolar rhabdomyosarcoma. Genomics. 2002;79(3):278–84. Epub 2002/02/28. pmid:11863357 doi: 10.1006/geno.2002.6703
[58]
Crepaldi T, Bersani F, Scuoppo C, Accornero P, Prunotto C, Taulli R, et al. Conditional activation of MET in differentiated skeletal muscle induces atrophy. J Biol Chem. 2007;282(9):6812–22. pmid:17194700 doi: 10.1074/jbc.m610916200
[59]
Takayama H, La Rochelle WJ, Anver M, Bockman DE, Merlino G. Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development. Proc Natl Acad Sci U S A. 1996;93(12):5866–71. pmid:8650184 doi: 10.1073/pnas.93.12.5866
[60]
Takayama H, LaRochelle WJ, Sharp R, Otsuka T, Kriebel P, Anver M, et al. Diverse tumorigenesis associated with aberrant development in mice overexpressing hepatocyte growth factor/scatter factor. Proc Natl Acad Sci U S A. 1997;94(2):701–6. pmid:9012848 doi: 10.1073/pnas.94.2.701
[61]
Dono R, Texido G, Dussel R, Ehmke H, Zeller R. Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice. EMBO J. 1998;17(15):4213–25. pmid:9687490 doi: 10.1093/emboj/17.15.4213
[62]
Mesbah K, Harrelson Z, Theveniau-Ruissy M, Papaioannou VE, Kelly RG. Tbx3 is required for outflow tract development. Circ Res. 2008;103(7):743–50. doi: 10.1161/CIRCRESAHA.108.172858. pmid:18723448
[63]
Furlan A, Roux B, Lamballe F, Conti F, Issaly N, Daian F, et al. Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures. PLoS One. 2012;7(10):e46738. Epub 2012/10/17. doi: 10.1371/journal.pone.0046738. pmid:23071625
[64]
Furlan A, Stagni V, Hussain A, Richelme S, Conti F, Prodosmo A, et al. Abl interconnects oncogenic Met and p53 core pathways in cancer cells. Cell Death Differ. 2011;18(10):1608–16. Epub 2011/04/02. doi: 10.1038/cdd.2011.23. pmid:21455220
[65]
Blitz E, Viukov S, Sharir A, Shwartz Y, Galloway JL, Pryce BA, et al. Bone ridge patterning during musculoskeletal assembly is mediated through SCX regulation of Bmp4 at the tendon-skeleton junction. Dev Cell. 2009;17(6):861–73. doi: 10.1016/j.devcel.2009.10.010. pmid:20059955
