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Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892, USA; and
* Department of Histology and Neurobiology, Dokkyo University School of Medicine, Mibu, Tochigi 321–02, Japan
2Correspondence: Laboratory of Molecular Embryology, Bldg. 18T, Rm. 106, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. E-mail: shi{at}helix.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis • extracellular matrix • Xenopus laevis • morphogenesis • organogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Many important cytokines,
morphogens, and growth factors are forced to pass through ECM in
order to reach their target cells. The nature of the local ECM
environment also affects cell migration during development.
Furthermore, cell–ECM contacts can directly affect cell fate
determination, e.g., apoptosis vs. proliferation and differentiation
(1
, 3
, 4)
. Thus, it is easy to envision that establishing
and maintaining a proper ECM environment plays a critical role in
organogenesis and tissue remodeling during development.
ECM remodeling is to a large extent mediated by matrix
metalloproteinases (MMPs), a family of
Zn2+-dependent proteinases that are capable of
degrading proteinaceous components of the ECM (5
6
7
8)
.
About 20 different MMPs have been identified in vertebrates, including
collagenases, gelatinases, stromelysins, and membrane-type MMPs, etc.
MMPs are secreted as inactive proenzymes, with the exception of
stromelysin-3 (ST3) and membrane-type MMPs (9
, 10)
, which
are activated intracellularly. The proenzymes are kept inactive due to
the formation of a fourth coordination bond with the catalytic
Zn2+ by a conserved cysteine residue in the
propeptide (11
12
13)
. The proenzymes can be activated in
the ECM or on the cell surface through the proteolytic removal of the
propeptide (12
13
14)
. Different activated MMPs have
distinct but overlapping substrate specificity, but collectively they
can degrade all proteinaceous components of the ECM (10
, 15)
. Thus, differential regulation of the levels of various MMPs
is expected to lead to distinct ECM remodeling events, resulting in
different biological consequences. In fact, MMP genes have been shown
to be regulated differentially in different developmental and
pathological processes, such as postlactation involution of the mammary
gland and cancer metastasis (16
17
18)
.
We are interested in the roles of different MMPs during development. We have chosen the South African toad Xenopus laevis as a model system. Xenopus laevis embryos develop externally and independent of maternal influence. Furthermore, like other anurans, they undergo a biphasic developmental process, first forming free-living tadpoles and subsequently metamorphosing into the adult form, which has a very different living style. Thus, it provides an opportunity to investigate MMP function during both embryogenesis and the subsequent postembryonic organ remodeling stages.
The involvement of MMPs in anuran development was first revealed more
than 30 years ago when Gross and colleagues first discovered
collagenase activities in the resorbing tail during metamorphosis
(19)
. More recently, several MMP genes were found to be
induced by thyroid hormone in various organs during metamorphosis
(20
21
22
23)
. These MMP genes appear to be under distinct
regulation by thyroid hormone receptors. We showed earlier that at
least two of the MMPs—the Xenopus laevis ST3 and
collagenase-4 (Col4)—are also expressed during embryonic development
(21
, 22)
. To investigate the possible roles of MMPs during
embryogenesis, we have now analyzed in detail the spatial temporal
expression profiles of ST3 and Col4 as well as another MMP,
collagenase-3 (Col3). By overexpressing thyroid hormone receptors in
embryos, we show that the MMPs are differentially affected, thus
providing in vivo evidence for the distinct regulation of
these MMPs during Xenopus development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TR) and 9-cis retinoic
acid receptor (RXR) cDNAs (24)
were linearized with
EcoRI, phenol/chloroform extracted twice, and ethanol
precipitated. One microgram of the DNA was transcribed in
vitro with a SP6 transcription kit (Ambion, Austin, Tex.). The
quantity and quality of the resulting RNA were assayed on a 1%
agarose-formaldehyde gel.
In vitro fertilization of Xenopus
eggs and embryo injections
Xenopus laevis females were primed and
ovulated eggs were fertilized by standard methods (25)
.
For embryo injection, the jelly coat was removed by washing the
fertilized eggs with 3% cysteine, pH 8.0. This was followed by four to
six washes with 0.1x MMR. Healthy looking embryos were collected just
after beginning the first division and immediately transferred to 0.5x
MMR, 2% Ficoll and injected in both sides with a total of 50–500 pg
of each of the TR and RXR mRNA (in 5 nl total volume)/embryo.
Control and injected embryos were kept in 0.5x MMR, 2% Ficoll for
4–6 h after injection and then transferred to 0.1x MMR. Embryos were
incubated without hormone or in the presence of 100 nM thyroid hormone
T3 (3, 5, 3'-triiodothyronine). The culture
medium was changed daily.
Northern blot hybridization
Five micrograms of total RNA from Xenopus embryos of
a given stage were electrophoresed on 1% agarose/formaldehyde gel and
transferred onto a GeneScreen membrane (NEN) after partial hydrolysis
with NaOH (26)
. Hybridization was done by using the cDNA
inserts of Xenopus stromelysin-3 (21)
,
collagenase-3 (23)
, and collagenase-4 (22)
.
After overnight hybridization at 42°C (in 50% formamide, 5x SSPE,
0.2% sodium dodecyl sulfate (SDS), 10% dextran sulfate, 5x
Denhardt’s solution, and 100 µg/ml denatured salmon sperm DNA), the
filters were washed three times for 5–10 min each at room temperature
in 2x SSC and 0.2% SDS. Stringent washes were then performed twice
for 25 min each in 0.25x SSC and 0.2% SDS at 65°C. To control for
RNA loading and quality, Northern blots were stained with methylene
blue before hybridization (27)
.
Whole-mount in situ hybridization
The protocol was essentially that of Harland (28)
.
Briefly, albino Xenopus embryos, staged according to
Neiuwkoop and Faber (29)
, were fixed with
paraformaldehyde, rinsed with PBS, and treated with proteinase K. After
refixation with paraformaldehyde, embryos were acetylated with acidic
anhydride and prehybridized in hybridization solution minus probe.
Full-length Xenopus ST3 (21)
, Col3
(23)
, and Col4 (22)
were subcloned into
Stratagene pBluescript II plasmid, linearized with appropriate
restriction enzymes, and transcribed with T3 or T7 RNA polymerase.
Digoxigenin-labeled antisense and control sense RNA probes were
generated using a Boehringer Mannheim Genius 4 kit. Hybridization was
carried out at 60°C overnight, followed by high-stringency washes of
0.2% SSC, 0.1% CHAPS at 60°C. An alkaline phosphatase-conjugated
digoxigenin antibody was used and the calorimetric reaction developed
using NBT and BCIP. Embryos were then postfixed in Bouin’s, dehydrated
with methanol, and cleared with benzyl benzoate and benzyl alcohol for
photography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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22
23)
. Their
expression profiles during metamorphosis suggest they are involved in
larval tissue resorption and adult organogenesis. As anurans such as
Xenopus undergo a biphasic developmental process, i.e.,
embryogenesis to form aquatic tadpoles and subsequent metamorphosis to
form (often terrestrial) frogs, we wanted to determine whether these
MMPs were also involved in embryonic organogenesis. Thus, we analyzed
their spatial and temporal expression profiles during
Xenopus embryogenesis.
To compare the developmental expression profiles of the three MMPs, we
isolated total RNAs from embryos or tadpoles at different stages of
development. Northern blot analysis of the RNA, followed by
densitometry showed that the three MMPs were expressed during
embryogenesis (Fig. 1
). Col3 was the first gene to be activated. Its mRNA was up-regulated
immediately after zygotic transcription beginning at stage 9/10, the
midblastula transition (Fig. 1)
. After reaching a peak at stage 40/41
(shortly after tadpole hatching [stage 35/36]), Col3 mRNA levels
decreased to a minimum in premetamorphic tadpoles and were up-regulated
again to high levels during metamorphosis (stages 54–66).
|
Unlike Col3, Col4 mRNA level was low throughout development except
around stages 44/45, when a peak level was detected (Fig. 1)
coinciding
with the onset of tadpole feeding (29)
. During
metamorphosis, the Col4 mRNA level was low when analyzed in whole
animals, although it was up-regulated in some metamorphosing organs
such as the tail (22)
.
ST3 expression resembled that of Col3 in that both had two peak levels
of expression: one at late embryogenesis and the other at metamorphic
climax. The embryonic expression of both genes correlated with a period
of extensive tissue remodeling, cell migration, and morphogenesis
(29
, 30)
. On the other hand, ST3 was activated much later
than Col3, with its mRNA level noticeably up-regulated around neurula
stages (stages 16/17). In addition, the down-regulation of ST3 mRNA
levels occurred after stage 33/34, earlier than those of Col3 and Col4.
The reactivation of Col3 and ST3 in postembryonic tadpoles paralleled
the rise in endogenous plasma levels of thyroid hormone
(31)
, which initiated the metamorphic process (32
, 33)
. As metamorphosis approached completion, the mRNA levels of
both genes were again down-regulated. This indicates that these MMPs
participate in tissue remodeling and organogenesis during both
embryogenesis and metamorphosis.
Contrasting spatial distribution patterns of MMP mRNAs during
embryogenesis
Whole-mount in situ hybridization was then carried on
albino Xenopus embryos to determine the spatial expression
profiles of Col3, Col4, and ST3. In agreement with Northern blot
analysis, ST3 mRNA was not detected until neurula stages (see stage 18,
Fig. 2A
), when it was expressed in newly forming mesodermal and
ectodermal axial structures. As development progresses toward the tail
bud stage, ST3 expression was most abundant in two regions, the head
and posterior regions, of the stage 32 embryo (Fig. 2C
). In
the head region, ST3 mRNA was expressed in three distinct small domains
at the proximal edges of the three pharyngeal folds. There were also
low levels of ST3 expression in several head structures, particularly
the eyes and the otic vesicles (Fig. 2C
). Shortly after this
stage, ST3 expression in this region decreased and became more diffuse
(data not shown). In the posterior region of the stage 32 embryo, ST3
was expressed at the posterior margin of the endoderm at the back of
the embryo. This narrow band of expression extended anteriorly along
the border with the lateral mesoderm (just below the somites).
When viewed dorsally at stage 35/36 (Fig. 2E
), this anterior
extending band of expression appeared to be highly patterned.
Expression along this posterior margin of the endoderm proceeded
anteriorly as the endoderm was brought forward during development (data
not shown). In later stages (about stage 45), as the intestine matures
and the tadpole initiates feeding (29)
, expression in this
dorsal region was greatly decreased (data not shown) and only a weak,
diffuse expression remained in the head, particularly in the region of
the future Meckel’s cartilage (Fig. 2F
).
|
Whole-mount in situ hybridization confirmed that Col3 was
expressed early in development. Strong expression was detected in the
ectoderm and in developing axial structures. At about stage 20 (Fig. 2B
), Col3 mRNA was abundant in the head and along the entire
dorsal axis. Shortly thereafter, at about stage 25, the axial
expression within the somites becomes segmented (see Fig. 2D
for a stage 32 embryo) and remained segmented until about stage 40
(data not shown). Expression along the axis was within the somites and
neural tube, but (somewhat surprisingly) not the notochord, which is
surrounded by a collagen sheath (Fig. 2D
, insert),
suggesting that Col3 is involved in collagen degradation for somite
morphogenesis while leaving the collagen sheath surrounding the
notochord intact. By stage 45, there was little expression of Col3
along the dorsal axis, although expression was still detectable within
the head, particularly one of the branchial arches (Fig. 2G
).
Unlike ST3 or Col3 but consistent with the Northern blot data in Fig. 1
, Col4 was only transiently detectable, at about stage 45, in the
ventral head structure (Fig. 2H
, branchial arch) in an area
where much remodeling of mouth structures was occurring as feeding was
beginning. These results indicate that these three MMPs not only differ
in their temporal involvement in embryogenesis, but also play distinct
roles in the morphogenesis/remodeling of different organs/regions of
the embryo.
Differential regulation of MMP genes by thyroid hormone receptors
Although all three MMP genes are thyroid hormone
(T3) response genes, their regulation by
T3 differs. ST3 appears to be a direct
T3 response gene as its regulation by
T3 is fast, within a few hours of treatment of
premetamorphic tadpoles with physiological concentrations of
T3. Furthermore, its up-regulation occurs even
when protein synthesis inhibitors are present to block new protein
synthesis (23)
. On the other hand, both Col3 and Col4 are
likely to be indirectly regulated by T3 due to
their slow response to T3 treatment of
premetamorphic tadpoles (22
, 23)
.
In addition, all three MMP genes are down-regulated after stage 44/45
(Fig. 3
), when tadpole feeding begins. This coincides with the activation of
thyroid hormone and RXR, a heterodimerization partner of TR
genes (24
, 34)
. As unliganded TR/RXR heterodimers
function as transcriptional repressors on
T3-inducible genes (35
, 36)
, they
may participate directly or indirectly in the repression of these MMP
genes, particularly the ST3 gene.
|
Since ST3 and Col3 are expressed during embryogenesis when TR genes are
not expressed, the embryos offer an opportunity to investigate whether
TR/RXR directly regulates these MMPs. We overexpressed TR/RXR in
developing embryos by microinjecting their mRNAs into fertilized eggs.
Overexpression of TR/RXR led to embryonic deformations in the presence
of T3 (Figs. 3
, 4)
while causing relatively minor
but different defects in the absence of T3 (data
not shown; see also ref 37
). Overexpression of TR/RXR in
the absence of T3 had no detectable alterations
in the spatial distribution of Col3 and ST3 mRNAs (data not shown).
However, a small down-regulation of ST3 mRNA level was detected by
Northern blot analysis of the total RNA from 2-day-old embryos
microinjected with 500 pg each of the TR and RXR mRNAs
(37)
.
|
When the injected embryos were maintained in the presence of 100 nM
T3, the spatial distribution of ST3 was
drastically altered. At stage 16, when ST3 gene was just activated, ST3
mRNA was restricted to the developing axis in control embryos with or
without continuous T3 treatment (Fig. 3A
and data not shown). In contrast,
T3 treatment of embryos injected with 500 pg of
each of TR and RXR mRNAs led to aberrant expression of ST3 mRNA
as axial structures became perturbed (compare Figs. 3A
and
B
). In particular, the ST3 mRNA signal in what is likely the
future tail bud region (see below) became much more prominent
(arrowheads, Fig. 3B
) compared to that in control embryos.
Most of the embryos injected with 500 pg of TR/RXR mRNA failed to
develop much further than neurula stages in the presence of 100 nM
T3. Of the ones that survived to the stage when
control embryos reached stage 32, all displayed severe developmental
anomalies, particularly by axial defects (Fig. 4
). Similar to that seen in early embryos (Fig. 3B
), ST3 mRNA
expression was found to be restricted to the tail bud and head regions
(Fig. 4C
, D
), just like that in control embryos (Fig. 4A
). However, spatially ST3 expression was drastically
expanded in the tail bud region and to a lesser extent the head region
as well (compare Fig. 4A
with panels C, D for two
different mRNA-injected embryos). This expanded expression in the tail
bud region, as well as the aberrant ST3 expression in stage 16 embryos
(Fig. 3B
), may account for the up-regulation of ST3 mRNA
levels in the embryos as determined by Northern blot hybridization
(37)
. This expansion of ST3 expression was evident even at
low levels of TR/RXR overexpression (with only 50 pg of each mRNA
injected per embryo), which caused relatively minor anomalies (Fig. 4B
).
In parallel, we examined the expression of Col3 gene (we did not
analyze the Col4 gene as normally it is not expressed except around
stage 45, a stage when few embryos injected with 500 pg of mRNA
survived). Again, we found that in the absence of
T3, overexpression of TR/RXR with an injection of
either 50 or 500 pg of TR/RXR mRNA had little effect on Col3 mRNA
levels or spatial distribution (data not shown). In the presence of 100
nM T3, embryos with 500 pg of injected TR and
RXR mRNAs had severe axial defects and altered Col3 mRNA
distribution even at stage 12 (compare Fig. 3D
and
C, where the control embryo showed extensive Col3 expression
within the extending mesoderm and in many ectodermal cells). Unlike
ST3, Col3 expression appeared to be down-regulated by
T3 plus TR/RXR overexpression. This
down-regulation was more evident in older embryos (compare Figs. 4G
, H
with the control embryo in Fig. 4E
), but
Col3 mRNA remained largely restricted to dorsal axial structures.
Furthermore, this reduction in Col3 expression appeared to be
correlated with the reduced axial structure differentiation. At low
levels of TR/RXR overexpression (with 50 pg of each injected per
embryo), the Col3 mRNA levels and spatial distribution appeared to be
largely unaltered (Fig. 4F
), correlating with the
relatively minor defects in axial development. Thus, ST3 and Col3 are
not only expressed in different regions of the embryos, but also are
regulated differentially by TR/RXR in a manner consistent with ST3
being a direct T3 response gene whereas Col3 is
an indirect T3 response gene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 15)
, differential
expression of this family of ECM-degrading extracellular proteases can
selectively alter cell–ECM interactions, thus causing distinct
developmental and pathological effects. However, relatively little is
known about the roles of MMPs during embryogenesis. Although a number
of knockout and transgenic mice have been generated for several MMPs,
only limited information is obtained with regard to roles of MMPs in
development (38)
, possibly due to redundancy in MMP
function. We have shown here that three frog MMP genes have distinct
spatial and temporal expression profiles during Xenopus
embryogenesis. Furthermore, using the embryos as a model, we have
provided in vivo evidence for a differential function of
thyroid hormone receptors in the regulation of the MMPs during
development.
Thyroid hormone-dependent and independent regulation of MMP genes
Like other anurans, Xenopus embryogenesis takes
place in the absence of thyroid hormone whereas metamorphosis is
absolutely dependent on thyroid hormone. Our data here, together with
our earlier observations, indicate that all three Xenopus
MMPs are involved in both developmental processes (21
, 22)
. Furthermore, the MMP genes are up-regulated by thyroid
hormone during metamorphosis (21
22
23)
, whereas their
activation during embryogenesis occurs prior to the synthesis of
endogenous thyroid hormone around stage 53/54 (31)
. In
addition, all three MMPs are repressed after tadpole feeding begins at
stage 45, when TR and RXR genes are expressed, whereas thyroid
hormone is not yet available. It has been suggested that TR/RXR
heterodimers may serve to repress thyroid hormone response gene in
tadpoles prior to metamorphosis (39)
.
Despite the above similarities, the three MMP genes are under different
controls during frog development. Both in embryos and metamorphosing
tadpoles, ST3, Col3, and Col4 have distinct, although sometimes
overlapping, spatial and temporal expression profiles. Col3 and Col4
appear to be indirectly regulated by TR during metamorphosis (22
, 23)
. In contrast, ST3 is an immediate-early thyroid hormone
response gene. However, a thyroid response element has yet to be
identified in the ST3 gene (40)
. By overexpressing TR/RXR
in early embryos, we have shown here that ST3 expression is
up-regulated by the overexpressed T3-bound TR/RXR
and that its area of expression is also expanded (Figs. 3
, 4)
. On the
other hand, the T3-bound TR/RXR in early embryos
reduces Col3 mRNA levels, in contrast to that observed during
metamorphosis. This supports the view that the regulation of Col3 is
likely an indirect effect caused by TR/RXR-induced developmental
anomalies. These results provide in vivo evidence that
TR/RXR directly regulates ST3 expression. Although we failed to observe
significant repression of ST3 by unliganded TR/RXR with whole-mount
in situ hybridization, this was possibly due to insufficient
levels of TR/RXR overexpression. Thus, it is tempting to speculate that
the earlier down-regulation of ST3 in tadpoles (after stage 33/34) is
due to direct repression of ST3 gene by unliganded TR/RXR, whereas the
eventual reduction in Col3 and Col4 repression in postfeeding tadpoles
is likely an indirect effect of the expression of unliganded TR/RXR in
premetamorphic tadpoles. Furthermore, the up-regulation of ST3 by
exogenous, T3-bound TR/RXR is likely to
contribute to the drastic defects of such embryos.
Spatially and temporally restricted roles of MMPs during
embryogenesis
As stated earlier, all three MMPs have two periods of expression,
one during embryogenesis and the other during metamorphosis. This is
not surprising, since both periods involve extensive tissue remodeling
as opposed to the intervening premetamorphic stages, when tadpole
growth predominates.
ST3 expression is tightly regulated during embryogenesis. ST3 mRNA is
present in areas of active morphogenesis (e.g., head structures such as
the pharyngeal folds and eyes, etc.), but absent from other actively
developing areas such as the heart region. The head and the pharyngeal
arches, which will give rise to the mandibular, hyoid, and branchial
arches, involve massive neural crest cell migration (41)
.
In the eyes and otic vesicles, in addition to tissue remodeling,
extensive inductive signaling events occur as cell identities are
established in these organs. Thus, as an MMP, ST3 may participate in
these morphogenetic events whereas it is absent in the axial structures
(neural tube, notochord, somites, etc.) where most large structural ECM
molecules such as collagens are found. ST3 expression in the branchial
arches coincides with high levels of apoptotic cells in this region at
these stages of development (42)
In the dorsal tissues, ST3 expression is largely restricted to the
tailing edge of endoderm as it is resorbed due to yolk utilization
during development. Again, it is an area where both remodeling and cell
death (removal of endodermal yolk cells) are taking place. In fact, a
whole-mount TUNEL analysis has shown that this region is rich in
apoptotic cells (42)
.
In the tail region of the developing embryo, there is also a distinct
pattern of ST3 expression beneath the dorsal most somites. Although no
particular structure has been ascribed to this area at such early
stages (stages 32–35), as development occurs and tail elongation and
ventral yolk regression continue, this area corresponds to the region
where future hind limb buds will develop at stage 45 (29)
,
implicating a role of ST3 in tissue patterning. Alternatively, as ST3
expression in the dorsal region is both at the dorsal most margin and
lateral to the dorsal somites, it may be related to the remodeling
events that occur during tail elongation, hindgut and proctodeum
formation, when numerous cell–cell interactions and cell migration
take place to facilitate dorsal structure formation (43
, 44)
.
In contrast to ST3, Col3 gene is activated earlier than ST3 or Col4 and
is abundantly expressed in many tissues, most prominently in axial
structures at the time when early rapid axis elongation takes place.
Col3 is expressed in somites through most of embryogenesis.
Xenopus somites are somewhat unique in that they establish
their final pattern and orientation through the rotation of blocks of
large mesodermal cells (45)
. The presence of Col3 in the
early embryos may facilitate this rotation by remodeling the ECM. In
addition, low levels of Col3 are expressed in the neural tube, which is
remodeled as it grows and is reinforced by collagen fibers, and around
the notochord, which is surrounded by collagen and needs to be
remodeled as it elongates. These results suggest that little collagen
degradation is needed in these areas or that additional,
yet-unidentified collagenases may be involved in embryogenesis as well.
In the head, there is a transient overlap between Col3 and ST3
expression. This occurs in the pharyngeal arches of tail bud embryos,
although ST3 expression is more restricted. Later in development, ST3
is expressed primarily in Meckel’s cartilage, in agreement with the
findings in Xenopus by Berry et al. (46)
and in
mouse by Chin and Werb (47)
. In contrast, both Col3 and
Col4 are expressed in one of the branchial arches. This is not
surprising as there is extensive chondrogenesis occurring in the head
at late embryonic stages (about stage 45), particularly in the
branchial arches and in the forming mandible when extensive collagen is
being laid down as cartilage is formed in these structures. The
importance of collagenases at stage 45 is also highlighted by the
expression of Col4, which is abundantly expressed only at this stage.
In conclusion, we have shown here that ST3, Col3, and Col4 have distinct expression profiles during Xenopus embryogenesis. The regions of ST3 expression appear to be rich in apoptotic cells, suggesting that ST3 may participate in cell fate determination, likely through modification of the ECM. On the other hand, Col3 is likely to play a role in dorsal axial formation. All three MMPs appear to participate in the head development but are involved in different regions at different developmental stages. Finally, our overexpression studies provide in vivo evidence for a possible mechanism of the differential regulation of those MMP genes toward the end of embryogenesis and during metamorphosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication June 2, 1999. Revised for publication September 27, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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