We have investigated the role of proteinases in the developmental program of bone, cartilage, tongue muscle and epithelial differentiation and remodeling in the mandibular arch during murine embryogenesis. Expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) was tissue-specific with little or no expression in the epithelium of tooth buds, tongue or oral cavity. Gelatinase A mRNA transcripts were strongly expressed in the perichondrium of Meckel's cartilage and mesenchymal areas of embryonic day 13–15 mandibles, whereas gelatinase B, collagenase-3, TIMP-1 and TIMP-2 mRNA were found primarily in the ossifying areas of the mandibles. The skeletal muscle of the tongue expressed stromelysin-3, TIMP-2 and TIMP-3 mRNA while stromelysin-3, TIMP-2 and gelatinase A were seen in the overlying connective tissue layer. Gelatinase A, gelatinase B, stromelysin-1, urokinase, TIMP-1 and TIMP-2 mRNA and protein activities were also detected in cultured mandibular explants. Culture of day 10 mandibular explants with a hydroxamic acid metalloproteinase inhibitor, but not with inhibitors of metalloendopeptidases (thiorphan and phosphoramidon), serine proteinases (aprotinin), cysteine proteinases (leupeptin) and urokinase (amiloride), altered mandibular morphogenesis dramatically. Development of the tongue (glossogenesis) and cartilage, but not bone or teeth was affected. Formation of the oral sulcus and fusion of the two epithelia of the medial sulcus were inhibited, and number and migration of myoblasts decreased. The resulting ‘tongue-tied phenotype’ indicates that MMPs are involved in epithelial morphogenesis and the migration of myoblasts to the region of the tongue. Development of the anterior segment of Meckel's cartilage was also inhibited and proteoglycan content of the cartilage was reduced by inhibiting MMPs. Our data suggest that matrix metalloproteinases play a pivotal role in the morphogenesis of structures derived from epithelium (oral sulcus), cranial paraxial mesoderm (tongue) and cranial neural crest (Meckel's cartilage).

REFERENCES

REFERENCES
Adler
R. R.
,
Brenner
C. A.
,
Werb
Z.
(
1990
)
Expression of extracellular matrix-degrading metalloproteinases and metalloproteinase inhibitors is developmentally regulated during endoderm differentiation of embryonal carcinoma cells.
Development
110
,
211
220
Alexander
C. M.
,
Hansell
E. J.
,
Behrendtsen
O.
,
Flannery
M.
,
Kishnani
N. S.
,
Hawkes
S.
,
Werb
Z.
(
1996
)
Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation.
Development
122
,
1723
1736
Apte
S.
,
Hayashi
K.
,
Seldin
M.
,
Mattei
M.-G.
,
Hayashi
M.
,
Olsen
B.
(
1994
)
Gene encoding a novel murine tissue inhibitor of metalloproteinases (TIMP), TIMP-3, is expressed in developing mouse epithelia, cartilage, and muscle, and is located on mouse chromosome 10.
Dev. Dynam
200
,
177
197
Birkedal-Hansen
H.
,
Moore
W. G. I.
,
Bodden
M. K.
,
Windsor
L. J.
,
Birkedal-Hansen
B.
,
DeCarlo
A.
,
Engler
J. A.
(
1993
)
Matrix metalloproteinases: a review.
Critical Rev. Oral Biol. Med
4
,
197
250
Boone
T. C.
,
Johnson
M. J.
,
De Clerk
Y. A.
,
Langley
K. E.
(
1990
)
cDNA cloning and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases.
Proc. Nat. Acad. Sci.USA
87
,
2800
2804
Chai
Y.
,
Mah
A.
,
Crohin
C.
,
Groff
S.
,
Bringas
P.
,
Le
T.
,
Santos
V.
,
Slavkin
H. C.
(
1994
)
Specific transforming growth factor-subtypes regulate embryonic mouse Meckel's cartilage and tooth development.
Dev. Biol
162
,
85
103
Chin
J. R.
,
Murphy
G.
,
Werb
Z.
(
1985
)
Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase.
J. Biol. Chem
260
,
12367
12376
Collier
I. E.
,
Wilhelm
S. M.
,
Eisen
A. Z.
,
Marmer
B. L.
,
Grant
G. A.
,
Seltzer
J. L.
,
Kronberger
A.
,
He
C. S.
,
Bauer
E. A.
,
Goldberg
G. I.
(
1988
)
H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen.
J. Biol. Chem
263
,
6579
6587
D'Aquila
R. T.
,
Bechtel
L. J.
,
Videler
J. A.
,
Eron
K. K.
,
Gorczyca
P.
,
Kaplan
J. C.
(
1991
)
Maximizing sensitivity and specificity of PCR by pre-amplification heating.
Nucleic Acids Res
19
,
3749
–.
Deuchar
E. M.
(
1958
)
Experimental demonstration of tongue muscle origin in chick embryos.
J. Embryol. Exp. Morph
6
,
527
529
Flenniken
A. M.
,
Williams
B. R. G.
(
1990
)
Developmental expression of the endogenous TIMP gene and a TIMP-lacZ fusion gene in transgenic mice.
Genes Dev
4
,
1094
1106
Frohman
M. A.
,
Boyle
M.
,
Martin
G. R.
(
1990
).
Isolation of the mouse Hox-2.9 gene; analysis of embryonic expression suggests that positional information along the anterior-posterior axis is specified by mesoderm.
Development
110
,
589
607
Frommer
J.
,
Margolies
M. R.
(
1971
)
Contribution of Meckel's cartilage to ossification of the mandible in mice.
J. Dent. Res
50
,
1260
1267
Gack
S.
,
Vallon
R.
,
Schmidt
J.
,
Grigoriadis
A.
,
Tuckermann
J.
,
Schenkel
J.
,
Weiher
H.
,
Wagner
E. F.
,
Angel
P.
(
1995
)
Expression of interstitial collagenase during skeletal development of the mouse is restricted to osteoblast-like cells and hypertrophic chondrocytes.
Cell Growth Diff
6
,
759
767
Gewert
D. R.
,
Coulombe
B.
,
Castelino
M.
,
Skup
D.
,
Williams
B. R. G.
(
1987
)
Characterization and expression of a murine gene homologous to human EPA/TIMP: a virus-induced gene in the mouse.
EMBO J
6
,
651
657
Grobelny
D.
,
Poncz
L.
,
Galardy
R. E.
(
1992
)
Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeroginosa elastase by peptide hydroxamic acids.
Biochem
32
,
7152
7154
Hall
B. K.
(
1991
)
Cellular interactions during cartilage and bone development.
J. Craniofac. Genet. Dev. Biol
11
,
238
250
Hall
B. K.
,
Ekanayake
S.
(
1991
)
Effects of growth factors on the differentiation of neural crest cells and neural crest cell-derivatives.
Int. J. Dev. Biol
35
,
367
387
Hazelton
R. D.
(
1970
)
A radiographic analysis of the migration and fate of cells derived from the occiptal somites in the chick embryo with specific reference to the development of the hypoglossal musculature.
J. Embryol. Exp. Morph
24
,
455
466
Helms
J.
,
Thaller
C.
,
Eichele
G.
(
1994
)
Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud.
Development
120
,
3267
3274
Hudson
C. D.
,
Shapiro
B. L.
(
1973
)
An autoradiographic study of deoxyribonucleic acid synthesis in embryonic rat palatal shelf epithelium with reference to the concept of programmed cell death.
Arch. Oral Biol
18
,
77
84
Imai
H.
,
Osumi-Yamashita
O.
,
Youichirou
N.
,
Eto
K.
(
1996
)
Contribution of early-emigrating midbrain crest cells to the dental mesenchyme of mandibular molar teeth in rat embryos.
Dev. Biol
176
,
151
165
Kollar
E. J.
,
Mina
M.
(
1991
)
Role of the early epithelium in the patterning of the teeth and Meckel's cartilage.
J. Craniofac. Genet. Dev. Biol
11
,
223
228
Lefebvre
O.
,
Wolf
C.
,
Limacher
J.-M.
,
Hutin
P.
,
Wendling
C.
,
LeMeur
M.
,
Basset
P.
,
Rio
M.-C.
(
1992
)
The breast cancer-associated stromelysin-3 gene is expressed during mouse mammary gland apoptosis.
J. Cell Biol
119
,
997
1002
Librach
C. L.
,
Werb
Z.
,
Fitzgerald
M. L.
,
Chiu
K.
,
Corwin
N. M.
,
Esteves
R. A.
,
Grobelny
D.
,
Galardy
R.
,
Damsky
C. H.
,
Fisher
S. J.
(
1991
)
92-kD type IV collagenase mediates invasion of human cytotrophoblasts.
J. Cell Biol
113
,
437
449
Mattot
V.
,
Raes
M. B.
,
Henriet
P.
,
Eeckhout
Y.
,
Stehelin
D.
,
Vandenbunder
B.
,
Desbiens
X.
(
1995
)
Expression of interstitial collagenase is restricted to skeletal tissue during mouse embryogenesis.
J. Cell Sci
108
,
529
535
Mayo
M. L.
,
Bringas
P.
Jr.
,
Santos
V.
,
Shum
L.
,
Slavkin
H. C.
(
1992
)
Desmin expression during early mouse tongue morphogenesis.
Int. J. Dev. Biol
36
,
255
263
McClearn
D.
,
Medville
R.
,
Noden
D.
(
1995
)
Muscle cell death during development of head and neck muscles in the chick embryo.
Dev. Dynam
202
,
365
377
Miyake
T.
,
Cameron
A. M.
,
Hall
B. K.
(
1996
)
Stage-specific onset of condensation and matrix deposition for Meckel's and other first arch cartilages in inbred C57BL/6 mice.
J. Craniofac. Genet. Dev. Biol
16
,
32
47
Noden
D.
(
1986
)
Origins and patterning of craniofacial mesenchymal tissues.
J. Craniofac. Genet. Dev. Biol
2
,
15
31
Noden
D.
(
1991
)
Cell movements and control of patterned tissue assembly during craniofacial development.
J. Craniofac. Genet. Dev. Biol
11
,
192
213
Rappolee
D. A.
,
Wang
A.
,
Mark
D.
,
Werb
Z.
(
1989
)
Novel method for studying mRNA phenotypes in single or small numbers of cells.
J. Cell Biol
39
,
1
11
Reponen
P.
,
Sahlberg
C.
,
Huhtala
P.
,
Hurskainen
T.
,
Thesleff
I.
,
Tryggvason
K.
(
1992
)
Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development in degradation in human articular cartilage.
J. Biol. Chem
267
,
7856
7862
Reponen
P.
,
Sahlberg
C.
,
Munaut
C.
,
Thesleff
I.
,
Tryggvason
K.
(
1994
)
High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development.
J. Cell Biol
124
,
1091
1102
Sharpe
P. M.
,
Ferguson
M. W. J.
(
1988
)
Mesenchymal influences on epithelial differentiation in developing systems.
J. Cell Sci. (Suppl.)
10
,
195
230
Shum
L.
,
Sakakura
Y.
,
Bringas
P.
Jr.
,
Luo
W.
,
Snead
M. L.
,
Mayo
M.
,
Crohin
C.
,
Millar
S.
,
Buckley
S.
,
Hall
F. L.
,
Warburton
D.
,
Slavkin
H. C.
(
1993
)
EGF abrogation-induced fusilli-form dysmorphogenesis of Meckel's cartilage during embryonic mouse mandibular morphogenesis in vitro.
Development
118
,
903
917
Slavkin
H. C.
,
Bringas
P.
Jr.
,
Sasano
Y.
,
Mayo
M.
(
1989
)
Early embryonic mouse mandibular morphogenesis and cytodifferentiation in serumless, chemically defined medium: a model for studies of autocrine and/or paracrine regulatory factors.
J. Craniofac. Genet. Dev. Biol
9
,
185
205
Slavkin
H. C.
(
1990
)
Regulatory issues during early craniofacial development: a summary.
Cleft Palate J
27
,
101
109
Spencer-Dene
B.
,
Thorogood
P.
,
Nair
S.
,
Kenny
A. J.
,
Harris
M.
,
Henderson
B.
(
1994
)
Distribution of, and a putative role for, the cell-surface neutral metallo-endopeptidases during mammalian craniofacial development.
Development
120
,
3213
3226
Sympson
C. J.
,
Talhouk
R. S.
,
Alexander
C. M.
,
Chin
J. R.
,
Clift
S. M.
,
Bissell
M. J.
,
Werb
Z.
(
1994
)
Targeted expression of stromelysin-1 provides evidence for a role of proteinases in branching morphogenesis and the requirement for an intact basement membrane for tissue-specific gene expression.
J. Cell Biol
125
,
681
693
This content is only available via PDF.