Histochemistry and Cell Biology

, Volume 138, Issue 4, pp 557–571 | Cite as

Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling

  • Nikolaos Gkantidis
  • Christos Katsaros
  • Matthias ChiquetEmail author
Original Paper


Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5–15.5, we optimized and validated a simple double-labeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel’s cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo.


Mouse embryo Craniofacial development Matrix metalloproteinase Basement membrane In situ zymography Dye-quenched gelatin Immunofluorescence 



We thank Susan Blumer for valuable suggestions and excellent technical support, Sabrina Ruggiero for initial help with SDS-gel zymography, Jean-François Spetz for providing us with mouse embryos, and Neha Gadhari for critical reading of the manuscript.

Supplementary material

418_2012_982_MOESM1_ESM.pdf (3.4 mb)
(PDF 3449 kb)


  1. Abiko Y, Kutsuzawa M, Kowashi Y, Kaku T, Tachikawa T (1999) In situ detection of gelatinolytic activity in developing craniofacial tissues. Anat Embryol (Berl) 200:283–287CrossRefGoogle Scholar
  2. Aiken A, Khokha R (2010) Unraveling metalloproteinase function in skeletal biology and disease using genetically altered mice. Biochim Biophys Acta 1803:121–132PubMedCrossRefGoogle Scholar
  3. Al Aqeel A, Al Sewairi W, Edress B, Gorlin RJ, Desnick RJ, Martignetti JA (2000) Inherited multicentric osteolysis with arthritis: a variant resembling Torg syndrome in a Saudi family. Am J Med Genet 93:11–18PubMedCrossRefGoogle Scholar
  4. Aufderheide E, Ekblom P (1988) Tenascin during gut development: appearance in the mesenchyme, shift in molecular forms, and dependence on epithelial-mesenchymal interactions. J Cell Biol 107:2341–2349PubMedCrossRefGoogle Scholar
  5. Baker AH, Edwards DR, Murphy G (2002) Metalloproteinase inhibitors: biological actions and therapeutic opportunities. J Cell Sci 115:3719–3727PubMedCrossRefGoogle Scholar
  6. Blavier L, Lazaryev A, Groffen J, Heisterkamp N, DeClerck YA, Kaartinen V (2001) TGF-beta3-induced palatogenesis requires matrix metalloproteinases. Mol Biol Cell 12:1457–1466PubMedGoogle Scholar
  7. Bruni-Cardoso A, Lynch CC, Rosa-Ribeiro R, Matrisian LM, Carvalho HF (2010) MMP-2 contributes to the development of the mouse ventral prostate by impacting epithelial growth and morphogenesis. Dev Dyn 239:2386–2392PubMedCrossRefGoogle Scholar
  8. Butler GS, Overall CM (2009) Updated biological roles for matrix metalloproteinases and new “intracellular” substrates revealed by degradomics. Biochemistry 48:10830–10845PubMedCrossRefGoogle Scholar
  9. Chin JR, Werb Z (1997) Matrix metalloproteinases regulate morphogenesis, migration and remodeling of epithelium, tongue skeletal muscle and cartilage in the mandibular arch. Development 124:1519–1530PubMedGoogle Scholar
  10. Chiquet M, Eppenberger HM, Turner DC (1981) Muscle morphogenesis: evidence for an organizing function of exogenous fibronectin. Dev Biol 88:220–235PubMedCrossRefGoogle Scholar
  11. Clark IM, Young DA, Rowan AD (2010) Matrix metalloproteinase protocols. Humana Press, New YorkCrossRefGoogle Scholar
  12. Deryugina EI, Quigley JP (2006) Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev 25:9–34PubMedCrossRefGoogle Scholar
  13. Egeblad M, Shen HC, Behonick DJ, Wilmes L, Eichten A, Korets LV, Kheradmand F, Werb Z, Coussens LM (2007) Type I collagen is a genetic modifier of matrix metalloproteinase 2 in murine skeletal development. Dev Dyn 236:1683–1693PubMedCrossRefGoogle Scholar
  14. Ehrismann R, Chiquet M, Turner DC (1981) Mode of action of fibronectin in promoting chicken myoblast attachment. Mr = 60,000 gelatin-binding fragment binds native fibronectin. J Biol Chem 256:4056–4062PubMedGoogle Scholar
  15. Evans RD, Itoh Y (2007) Analyses of MT1-MMP activity in cells. Methods Mol Med 135:239–249PubMedCrossRefGoogle Scholar
  16. Frederiks WM, Mook OR (2004) Metabolic mapping of proteinase activity with emphasis on in situ zymography of gelatinases: review and protocols. J Histochem Cytochem 52:711–722PubMedCrossRefGoogle Scholar
  17. Galis ZS, Sukhova GK, Libby P (1995) Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue. FASEB J 9:974–980PubMedGoogle Scholar
  18. Hibbs MS, Hoidal JR, Kang AH (1987) Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages. J Clin Invest 80:1644–1650PubMedCrossRefGoogle Scholar
  19. Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, Mankani M, Robey PG, Poole AR, Pidoux I, Ward JM, Birkedal-Hansen H (1999) MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 99:81–92PubMedCrossRefGoogle Scholar
  20. Iamaroon A, Wallon UM, Overall CM, Diewert VM (1996) Expression of 72-kDa gelatinase (matrix metalloproteinase-2) in the developing mouse craniofacial complex. Arch Oral Biol 41:1109–1119PubMedCrossRefGoogle Scholar
  21. Ikeda M, Maekawa R, Tanaka H, Matsumoto M, Takeda Y, Tamura Y, Nemori R, Yoshioka T (2000) Inhibition of gelatinolytic activity in tumor tissues by synthetic matrix metalloproteinase inhibitor: application of film in situ zymography. Clin Cancer Res 6:3290–3296PubMedGoogle Scholar
  22. Knauper V, Cowell S, Smith B, Lopez-Otin C, O’Shea M, Morris H, Zardi L, Murphy G (1997) The role of the C-terminal domain of human collagenase-3 (MMP-13) in the activation of procollagenase-3, substrate specificity, and tissue inhibitor of metalloproteinase interaction. J Biol Chem 272:7608–7616PubMedCrossRefGoogle Scholar
  23. Koshikawa N, Giannelli G, Cirulli V, Miyazaki K, Quaranta V (2000) Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5. J Cell Biol 148:615–624PubMedCrossRefGoogle Scholar
  24. Kudo T, Takino T, Miyamori H, Thompson EW, Sato H (2007) Substrate choice of membrane-type 1 matrix metalloproteinase is dictated by tissue inhibitor of metalloproteinase-2 levels. Cancer Sci 98:563–568PubMedCrossRefGoogle Scholar
  25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  26. Lambert V, Wielockx B, Munaut C, Galopin C, Jost M, Itoh T, Werb Z, Baker A, Libert C, Krell HW, Foidart JM, Noel A, Rakic JM (2003) MMP-2 and MMP-9 synergize in promoting choroidal neovascularization. FASEB J 17:2290–2292PubMedGoogle Scholar
  27. Lombardi F, Fasciglione GF, D’Apice MR, Vielle A, D’Adamo M, Sbraccia P, Marini S, Borgiani P, Coletta M, Novelli G (2008) Increased release and activity of matrix metalloproteinase-9 in patients with mandibuloacral dysplasia type A, a rare premature ageing syndrome. Clin Genet 74:374–383PubMedCrossRefGoogle Scholar
  28. Martignetti JA, Aqeel AA, Sewairi WA, Boumah CE, Kambouris M, Mayouf SA, Sheth KV, Eid WA, Dowling O, Harris J, Glucksman MJ, Bahabri S, Meyer BF, Desnick RJ (2001) Mutation of the matrix metalloproteinase 2 gene (MMP2) causes a multicentric osteolysis and arthritis syndrome. Nat Genet 28:261–265PubMedCrossRefGoogle Scholar
  29. Merkle M, Ribeiro A, Sauter M, Ladurner R, Mussack T, Sitter T, Wornle M (2010) Effect of activation of viral receptors on the gelatinases MMP-2 and MMP-9 in human mesothelial cells. Matrix Biol 29:202–208PubMedCrossRefGoogle Scholar
  30. Miettinen PJ, Chin JR, Shum L, Slavkin HC, Shuler CF, Derynck R, Werb Z (1999) Epidermal growth factor receptor function is necessary for normal craniofacial development and palate closure. Nat Genet 22:69–73PubMedCrossRefGoogle Scholar
  31. Mosig RA, Dowling O, DiFeo A, Ramirez MC, Parker IC, Abe E, Diouri J, Aqeel AA, Wylie JD, Oblander SA, Madri J, Bianco P, Apte SS, Zaidi M, Doty SB, Majeska RJ, Schaffler MB, Martignetti JA (2007) Loss of MMP-2 disrupts skeletal and craniofacial development and results in decreased bone mineralization, joint erosion and defects in osteoblast and osteoclast growth. Hum Mol Genet 16:1113–1123PubMedCrossRefGoogle Scholar
  32. Mott JD, Werb Z (2004) Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 16:558–564PubMedCrossRefGoogle Scholar
  33. Mungall BA, Pollitt CC, Collins R (1998) Localisation of gelatinase activity in epidermal hoof lamellae by in situ zymography. Histochem Cell Biol 110:535–540PubMedCrossRefGoogle Scholar
  34. Nagel H, Laskawi R, Wahlers A, Hemmerlein B (2004) Expression of matrix metalloproteinases MMP-2, MMP-9 and their tissue inhibitors TIMP-1, -2, and -3 in benign and malignant tumours of the salivary gland. Histopathology 44:222–231PubMedCrossRefGoogle Scholar
  35. Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM (2000) Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 18:1135–1149PubMedGoogle Scholar
  36. Nishida Y, Miyamori H, Thompson EW, Takino T, Endo Y, Sato H (2008) Activation of matrix metalloproteinase-2 (MMP-2) by membrane type 1 matrix metalloproteinase through an artificial receptor for ProMMP-2 generates active MMP-2. Cancer Res 68:9096–9104PubMedCrossRefGoogle Scholar
  37. Odaka C, Tanioka M, Itoh T (2005) Matrix metalloproteinase-9 in macrophages induces thymic neovascularization following thymocyte apoptosis. J Immunol 174:846–853PubMedGoogle Scholar
  38. Oh LY, Larsen PH, Krekoski CA, Edwards DR, Donovan F, Werb Z, Yong VW (1999) Matrix metalloproteinase-9/gelatinase B is required for process outgrowth by oligodendrocytes. J Neurosci 19:8464–8475PubMedGoogle Scholar
  39. Overall CM (2002) Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites. Mol Biotechnol 22:51–86PubMedCrossRefGoogle Scholar
  40. Parks WC, Wilson CL, Lopez-Boado YS (2004) Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol 4:617–629PubMedCrossRefGoogle Scholar
  41. Paulsson M, Aumailley M, Deutzmann R, Timpl R, Beck K, Engel J (1987) Laminin-nidogen complex. Extraction with chelating agents and structural characterization. Eur J Biochem 166:11–19PubMedCrossRefGoogle Scholar
  42. Porto IM, Rocha LB, Rossi MA, Gerlach RF (2009) In situ zymography and immunolabeling in fixed and decalcified craniofacial tissues. J Histochem Cytochem 57:615–622PubMedCrossRefGoogle Scholar
  43. Robbins JR, McGuire PG, Wehrle-Haller B, Rogers SL (1999) Diminished matrix metalloproteinase 2 (MMP-2) in ectomesenchyme-derived tissues of the Patch mutant mouse: regulation of MMP-2 by PDGF and effects on mesenchymal cell migration. Dev Biol 212:255–263PubMedCrossRefGoogle Scholar
  44. Sakakura Y, Hosokawa Y, Tsuruga E, Irie K, Yajima T (2007) In situ localization of gelatinolytic activity during development and resorption of Meckel’s cartilage in mice. Eur J Oral Sci 115:212–223PubMedCrossRefGoogle Scholar
  45. Sakuraba I, Hatakeyama J, Hatakeyama Y, Takahashi I, Mayanagi H, Sasano Y (2006) The MMP activity in developing rat molar roots and incisors demonstrated by in situ zymography. J Mol Histol 37:87–93PubMedCrossRefGoogle Scholar
  46. Shi J, Son MY, Yamada S, Szabova L, Kahan S, Chrysovergis K, Wolf L, Surmak A, Holmbeck K (2008) Membrane-type MMPs enable extracellular matrix permissiveness and mesenchymal cell proliferation during embryogenesis. Dev Biol 313:196–209PubMedCrossRefGoogle Scholar
  47. Snoek-van Beurden PA, Von den Hoff JW (2005) Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques 38:73–83PubMedCrossRefGoogle Scholar
  48. Stickens D, Behonick DJ, Ortega N, Heyer B, Hartenstein B, Yu Y, Fosang AJ, Schorpp-Kistner M, Angel P, Werb Z (2004) Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development 131:5883–5895PubMedCrossRefGoogle Scholar
  49. Trachslin J, Koch M, Chiquet M (1999) Rapid and reversible regulation of collagen XII expression by changes in tensile stress. Exp Cell Res 247:320–328PubMedCrossRefGoogle Scholar
  50. Visse R, Nagase H (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 92:827–839PubMedCrossRefGoogle Scholar
  51. Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z (1998) MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411–422PubMedCrossRefGoogle Scholar
  52. Wang J, Tsirka SE (2005) Neuroprotection by inhibition of matrix metalloproteinases in a mouse model of intracerebral haemorrhage. Brain 128:1622–1633PubMedCrossRefGoogle Scholar
  53. Wehrle B, Chiquet M (1990) Tenascin is accumulated along developing peripheral nerves and allows neurite outgrowth in vitro. Development 110:401–415PubMedGoogle Scholar
  54. Woessner JF Jr (1995) Quantification of matrix metalloproteinases in tissue samples. Methods Enzymol 248:510–528PubMedCrossRefGoogle Scholar
  55. Xu X, Wang Y, Lauer-Fields JL, Fields GB, Steffensen B (2004) Contributions of the MMP-2 collagen binding domain to gelatin cleavage. Substrate binding via the collagen binding domain is required for hydrolysis of gelatin but not short peptides. Matrix Biol 23:171–181PubMedCrossRefGoogle Scholar
  56. Yan SJ, Blomme EA (2003) In situ zymography: a molecular pathology technique to localize endogenous protease activity in tissue sections. Vet Pathol 40:227–236PubMedCrossRefGoogle Scholar
  57. Zamilpa R, Lopez EF, Chiao YA, Dai Q, Escobar GP, Hakala K, Weintraub ST, Lindsey ML (2010) Proteomic analysis identifies in vivo candidate matrix metalloproteinase-9 substrates in the left ventricle post-myocardial infarction. Proteomics 10:2214–2223PubMedCrossRefGoogle Scholar
  58. Zankl A, Pachman L, Poznanski A, Bonafe L, Wang F, Shusterman Y, Fishman DA, Superti-Furga A (2007) Torg syndrome is caused by inactivating mutations in MMP2 and is allelic to NAO and Winchester syndrome. J Bone Miner Res 22:329–333PubMedCrossRefGoogle Scholar
  59. Zhang HQ, Chang M, Hansen CN, Basso DM, Noble-Haeusslein LJ (2011) Role of matrix metalloproteinases and therapeutic benefits of their inhibition in spinal cord injury. Neurotherapeutics 8:206–220PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Nikolaos Gkantidis
    • 1
  • Christos Katsaros
    • 1
  • Matthias Chiquet
    • 1
    Email author
  1. 1.Department of Orthodontics and Dentofacial OrthopedicsUniversity of BernBernSwitzerland

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