Departament of Developmental Genetics and Molecular Physiology, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, México
* Author for correspondence (e-mail: covs{at}ibt.unam.mx)
Accepted 26 September 2003
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SUMMARY |
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Key words: Morphogenesis, Apoptosis, Cell migration, Mouse
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Introduction |
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The MEE is composed of a basal columnar cell layer covered by flat cells
that constitute the periderm. During shelf growth, MEE is histologically
undistinguishable from oral or nasal epithelium, but it acquires distinctive
features just prior to fusion. At the molecular level, the MEE region appears
defined by the expression of several genes such as Tgfb3
(Fitzpatrick et al., 1990),
Egfr (Brunet et al.,
1993
), Tgfa (Citterio
and Gaillard, 1994
) and Fos
(Yano et al., 1996
). It is
believed that periderm cells shed before fusion to allow intimate contact
between shelves (Fitchett and Hay,
1989
).
Epithelial-mesenchymal transformation (EMT) is considered relevant for MES
degeneration (Fitchett and Hay,
1989; Griffith and Hay,
1992
; Shuler et al.,
1991
). EMT stands for the transdifferentiation of packed
epithelial cells to more loose mesenchymal cells, a process that involves
basal lamina degradation (and dramatic changes in the cytoskeleton), and
cell-cell and cell-extracellular matrix interactions
(Boyer et al., 1996
). The
migratory capacity of mesenchymal cells allows them to move far from their
site of origin. Once the EMT process occurs, transdifferentiated cells can
give rise to specific cell types, just as neural crest cells do
(Duband et al., 1995
), or can
contribute to form structures such as the heart valves, which are derived from
endocardial cells (Markwald et al.,
1975
). In the case of secondary palate, transdifferentiated
mesenchymal cells would not have a specific function. Several lines of
evidence suggest that EMT actually occurs during palate shelf fusion
(Fitchett and Hay, 1989
;
Griffith and Hay, 1992
;
Martinez-Alvarez et al., 2000
;
Shuler et al., 1991
;
Shuler et al., 1992
). However,
because only few cells have been detected to have transdifferentiated and
because there is lack of quantitative analyses, other mechanisms for MES
degeneration should be considered. Migration of MEE cells towards the nasal
and oral regions has also been proposed to participate in shelf fusion
(Carette and Ferguson, 1992
).
Cell death, which has been known for many years to occur in the developing
palate (DeAngelis and Nalbandian,
1968
; Farbman,
1968
; Smiley and Dixon,
1968
), was until only recently implicated in MES degeneration
(Cuervo et al., 2002
;
Martinez-Alvarez et al., 2000
;
Mori et al., 1994
;
Taniguchi et al., 1995
). MES
degeneration could also result from a combination of cellular mechanisms such
as those described above.
The aim of the present work was to evaluate the relevance of EMT, epithelial cell migration and cell death in palate shelf fusion. Our results revealed a fundamental role of cell death in MES degeneration, without a significant contribution from EMT or basal MEE cell migration. However, we show data indicating that the ordered migration of periderm cells out from the basal MEE is necessary for normal shelf fusion. Furthermore, in contrast to the activation of cell death by the degradation of basal lamina (i.e. anoikis), we identified the activation of basal lamina degradation as a consequence of MEE cell death (`cataptosis').
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Materials and methods |
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Organ culture
Whole palates were cultured on filters floating on serum-free medium as
previously described (Cuervo et al.,
2002). We also developed a palate slice culture system based on
that reported by Knight et al. (Knight et
al., 1999
). Initially shelves of whole palates were allowed to
contact for 3 hours and were then embedded in 5% low-melting point agarose
(SeaPlaque GTG, FMC Bioproducts, Rockland, ME) in McCoy medium (Microlab,
México). Slices (200 µm) were obtained using a vibratome (Leica
VT1000S, Wetzlar, Germany) and collected in cold PBS (5.4 mM potassium
chloride, 138 mM sodium chloride, 22 mM glucose, 2 mM sodium-potassium
phosphate, pH 7.2). Slices were placed at the bottom of a 35 mm petri dish and
covered with a layer of 1% low-melting point agarose and 2 ml of McCoy medium.
At the end of culture, live slices were washed with PBS, fixed with 4%
paraformaldehyde and processed for TUNEL in wholemount
(Conlon et al., 1995
).
Cytochalasin D (6 µM; Sigma, St Louis, MO), cycloheximide (20 µg/ml;
Sigma, St Louis, MO), retinoic acid (20 µM; Sigma, St Louis, MO),
staurosporin (20 µM Sigma, St Louis, MO), BB3103 MMP inhibitor (10 µM;
British Biotech, Oxford, UK), or z-VAD (100 µM z-VAD; Biomol, Plymouth, PA)
were added directly to the culture medium. z-VAD anti-apoptotic activity has
also been tested in explant cultures of limbs undergoing interdigital
regression, and of developing spinal cords undergoing motoneuron degeneration.
Similarly, the inhibitory activity of BB3103 on metalloproteinases has been
tested by zymography using gelatin as substrate, and by the ability to avoid
the natural degradation of basal lamina in explant cultures containing
müllerian and wolfian ducts. All reagents remained in the medium for the
whole culture period.
Cell labeling
Complete MEE labeling was obtained by submerging whole palates in a 10
µM solution of 5-6-carboxy 2-7-dichlorofluorescein diacetate succinimidyl
ester (CCFSE; Molecular Probes, Eugene, OR) in PBS. Samples were incubated at
37°C for 15 minutes in the dye solution and washed twice with PBS before
culturing. Selective labeling of periderm cells with the same dye was attained
by a short incubation (30 seconds) of the palate explants in the same dye
solution at room temperature. Transfections with an adenovirus carrying the
lacZ reporter gene (Ad-lacZ) were carried out at 37°C
for 1.5 hours as previously described
(Cuervo et al., 2002).
Chimaeric palates were formed using one shelf from a CD1 embryo and the other
from an EGFP embryo. Shelf fusion under this condition took about 36 hours. In
all cases described above palates were cultured for different time periods,
fixed with 4% paraformaldehyde, embedded in paraffin or agarose, and sliced
for histology, immunohistochemistry or cell death detection.
Periderm cell removal
To remove superficial cells, palates were incubated in 0.25% trypsin
solution in Versene (Invitrogen, Grand Island, NY) at 4°C for 5 minutes.
After incubation, palate medial edges were extensively washed with the same
solution until the thin periderm cell layer came off. Samples were washed with
PBS and then incubated in DMEM containing 10% serum at room temperature for 10
minutes. Shelves were separated from the rest of the tissue and cultured on
filters. The same treatment was given to control palates except that wash with
0.25% trypsin was not performed.
Histology, immunofluorescence and TUNEL procedures
Cell death detection by the TUNEL method was performed using commercial
kits (Roche, Mannheim, Germany) or a whole-mount procedure as reported by
Conlon et al. (Conlon et al.,
1995). Immunohistochemistry for laminin (Rabbit anti-laminin,
Sigma, St. Louis, MO) was performed according to standard protocols. Except
for the Ad-lacZ labeling, labeled cells were detected by
epifluorescence (Eclipse TE 300, Nikon, Japan). Double detections combined
EGFP or fluorescein (CCFSE; in situ cell death detection kit, fluorescein,
Roche) with rhodamin (in situ cell death detection kit, rhodamin, Roche) or
Alexa-fluor 594 (goat anti-rabbit; Molecular Probes, Eugene, OR). Photographs
were taken with a digital camera (CoolSnap, Roper Scientific Inc., Trenton,
NJ).
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Results |
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We also followed the fate of MEE cells by forming chimaeric palates between
a wild-type shelf and a shelf from a mouse embryo that expresses
constitutively the GFP protein (WTEGFP). In this situation, the cells
that undergo EMT would be detected only if they cross the MES. That is,
transdifferentiated cells that migrated within the same shelf would not be
detected. Of course, in principle, mesenchymal cells present prior to shelf
contact could also migrate between shelves during or after fusion. No cells
were detected to cross the MES (Fig.
1A, WT
EGFP), supporting the low frequency of EMT occurrence
and also the limited migratory ability of mesenchymal cells from one shelf to
another. Regardless of the behavior of MEE and mesenchymal cells, nasal and
oral epithelial cells were observed to migrate between shelves
(Fig. 1B). From these
experiments, it was also interesting to observe that as the double adhered
epithelial layer turned into a single epithelial layer, intercalation of
epithelial cells from both shelves (i.e. EGFP-positive and EGFP-negative
cells) became obvious (Fig. 1A,
6-12 hours/WT
EGFP).
Fate of MEE cells in a palate slice culture system
In order to follow the fate of MEE cells continuously during MES
degeneration, we established a slice culture system in which cell migration
can be studied in further detail. MEE cells were labeled with CCFSE and
shelves were put in contact as described above. Three hours later, 200 µm
slices were obtained. At this time, MES appeared intact without signs of
degeneration (Fig. 2; 3 hours).
Next, slices were cultured as described in the Materials and methods. In
slices cultured for 3 hours (i.e. 6 hours after contact), the MES thinned; 6
hours later it seemed fragmented (Fig.
2; 6 hours and 12 hours, respectively). At this later time, no
labeled cells were found outside the MES. Twenty-four hours after contact, the
MES could not be visualized by phase-contrast microscopy, but fluorescent
cells were still detected within the fusion region
(Fig. 2, 24 hours). The great
majority of remaining cells at this time was of dying cells
(Fig. 2, TUNEL). Therefore, in
this culture system, MEE fusion takes place in the absence of detectable EMT
contribution.
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Migration of the periderm cells associated to the MEE
As previously shown, most MEE cells die during fusion. However, another
component associated to the MEE is the periderm that covers most of the shelf
surface that first comes into contact with the opposite shelf. In order to
label periderm cells preferentially, we stained palate shelves for a very
short period of time (i.e. 30 seconds). The selectivity of this staining
procedure can be clearly seen in slices of these preparations (see, for
example, Fig. 5, CCFSE/2
hours). To determine the fate of periderm cells, they were followed during the
fusion process. In contrast to MEE cells that appeared to die in situ,
periderm cells migrated to the oral and nasal ends of the MES, contributing to
the formation of the epithelial triangles, where most of them died
(Fig. 5, 2-8 hours).
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Discussion |
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Several years ago, Fitchett and Hay
(Fitchett and Hay, 1989)
presented the first evidence to suggest that EMT causes MES degradation.
Subsequently, other authors presented additional evidences supporting this
role of EMT (Kaartinen et al.,
1997
; Martinez-Alvarez et al.,
2000
; Shuler et al.,
1991
; Shuler et al.,
1992
). Currently, this idea prevails, in some cases neglecting the
participation of cell death (Young et al.,
2000
). In some of our experiments, we used experimental strategies
similar to those used in previous reports, but using an improved palate
culture system in which shelf fusion occurs within a similar time window as
observed in vivo. Under these conditions, we were unable to detect any obvious
participation of EMT. The few labeled cells found around the MES at the end of
culture were undergoing cell death (Table
1). Furthermore, when cell death was prevented, EMT was still not
detected, indicating that EMT does not compensate for the inability to
eliminate MEE by cell death. Because it is possible that individual
transdifferentiated cells escaped to our detection methods, we analyzed each
MEE cell within a 200 µm MES region using a novel slice culture system.
Again, we failed to detect any evidence of EMT and found, instead, that most
MEE cells were dying.
Why the discrepancy between our data and those previous reports? With some
exceptions (Sun et al., 1998),
very few MEE cells have been reported to undergo EMT, and in most reports
quantitative analyses are lacking. Moreover, in none of these reports, has it
been determined whether the assumed transdifferentiated cells are the dying
cells or whether they are phagocytes containing dying cells instead. These
drawbacks make it difficult to estimate the contribution of EMT to MES
degeneration. It is also possible that palate shelf fusion does not occur
equally along the rostrocaudal axis, which might explain the discrepancies if
the studies were performed at various points along this axis. We considered
this possibility and, thus, studied MES degeneration along the complete length
of the rostrocaudal axis. Another possible explanation for the contrasting
results obtained in our experiments is the use of an improved culture system.
Dissected shelves are usually put together and then cultured in the presence
of serum to allow fusion. We have found that our culture system allows a more
precise contact between shelves and an efficient fusion in the absence of
serum. Shuler et al. (Shuler et al.,
1992
) also labeled MEE in vivo with DiI and showed that clumps of
labeled cells remain around the fusion line. It is possible that artifactual
staining occurred in those experiments (DiI can easily precipitate), as
individual cells could not be visualized and DiI membrane incorporation would
not ensure the transfer of the dye to other more internal cells. Despite these
apparently conflicting results, we propose that cell death is the major
contributor to MES degeneration, even considering a low occurrence of EMT.
MEE cell migration has also been proposed as a mechanism for MES
degeneration (Carette and Ferguson,
1992). However, those studies did not take into account the
migration of the periderm cells that overlay the MEE. In our study, we stained
periderm cells preferentially and demonstrated that soon after contact they
migrate toward the oral and nasal cavities and form the epithelial triangles.
In keeping with this observation, when migration was blocked or periderm cells
were selectively eliminated, the epithelial triangles did not form. Shelf
fusion did take place in the absence of periderm cells, but it resulted in a
thinner palate. It has been considered that periderm cells shed before contact
(Fitchett and Hay, 1989
); our
data indicate, however, that epithelial triangles result from periderm cell
migration, a process that appears to be necessary for proper fusion.
Furthermore, a relevant finding of the present work was that periderm cell
migration plays role in activation of cell death of both periderm and basal
MEE cells. Periderm cells are likely to produce the filopodia and to be the
source of the proteoglycans required for shelf adhesion
(Gato et al., 2002
;
Taya et al., 1999
). We propose
that periderm cell migration is relevant for the efficient shelf fusion
regulating adhesion and cell death activation.
Anoikis is a term given to the process of cell death induced by the lack of
contact with the extracellular matrix. The basal lamina has been considered to
be an essential survival factor for epithelial cells in vitro and in vivo
(Coucouvanis and Martin, 1995;
Ruoslahti and Reed, 1994
). For
example, it has been shown that during mammary gland involution or Müller
duct regression, disrupting the underlying extracellular matrix induces
epithelial cell death (Pullan et al.,
1996
; Roberts et al.,
2002
). This prompted us to assess whether the trigger for MEE cell
death activation was basal lamina degradation. Blocking basal lamina
degradation by inhibiting MMP activity, however, had no effect on cell death.
These results contrast with two recent reports
(Blavier et al., 2001
;
Brown et al., 2002
) showing
partial or no MEE degeneration in the presence of the same MMP inhibitor used
here. In our experiments, we demonstrated [with high reproducibility even at
the low inhibitor dose (10 µM)] that the basal lamina was intact. In the
aforementioned reports, the integrity of basal lamina was not demonstrated,
and hence, their results can be interpreted as incomplete MEE degeneration.
Our observations in the presence of the MMP inhibitor do not imply that basal
lamina has no survival activity on MEE cells, but suggests that MEE cell death
is not triggered by basal lamina degradation. On the contrary, we found that
cell death activates basal lamina degradation (see
Fig. 4). To our knowledge, this
is an unprecedented finding that gives a new function to the process of cell
death. We propose the term `cataptosis' (a Greek word meaning downfall) to
describe this phenomenon. Cataptosis may occur in different developmental
process involving tissue regression, in order to coordinate cell degeneration
with extracellular matrix degradation. In the palate, the basal lamina
degradation activity is restricted to the dying MEE cells, suggesting that
specific factors give them this property (see below). This conclusion is also
in opposition to the participation of EMT, as inhibition of basal lamina
degradation can block EMT (Song et al.,
2000
), and MMPs can directly induce EMT
(Lochter et al., 1997
).
Collagen IV and laminin, the most abundant components of basal lamina, are
likely to be the major MMPs substrates during basal lamina degradation. MMPs
are found extracellularly and also bound to the plasma membrane (MTMMPs)
(Birkedal-Hansen, 1995). The
MMP activity could be regulated at different levels. MMP gene expression is
characteristic during tissue remodeling but postranslational regulation is
crucial for enzymatic activity. With the exception of MT-MMPs, MMPs are
synthesized as inactive proenzymes that need to be processed by other enzymes,
such as plasmin or other MMPs, to become active
(Nagase, 1997
). Furthermore,
MMP activity can be negatively regulated by direct binding of proteins such as
members of the tissue inhibitor of metalloproteinases (TIMP) family
(Gomez et al., 1997
). Among
the several MMPs and their inhibitors described to date, MT1-Mmp
(Mmp14 Mouse Genome Informatics), Mmp2, Mmp3, Mmp9,
Mmp13, Timp1 and Timp2 are expressed in the developing palate
(Blavier et al., 2001
;
Morris-Wiman et al., 2000
).
MT1-Mmp, Mmp13 and Timp2 are specifically expressed in the
MEE at the time fusion occurs (Blavier et
al., 2001
). These genes could be the ones that provide the MEE
with the distinct ability to activate cataptosis. MMP2 and MMP13 can digest
collagen IV (Knäuper et al.,
1997
), suggesting a role for these enzymes in basal lamina
degradation, although their role in vivo has not been demonstrated. TIMP2
could prevent extracellular matrix degradation; it has been shown, however,
that it is also relevant for the efficient MMP2 activation in vivo
(Wang et al., 2000
). MT1-MMP,
conversely, could be the initiator of a cascade of MMPs involved in
cataptosis. Recently, it was reported that MT1-MMP and MMP2 translocate from
the cytoplasm to the plasma membrane in endothelial cells upon stimulation of
apoptosis (Levkau et al.,
2002
). Furthermore, MT1-MMP can activate proMMP13
(Knäuper et al., 2002
).
However, as Mt1-Mmp null mutants do not have an obvious palate
phenotype (Holmbeck et al.,
1999
), the latter hypothesis would imply that redundant mechanisms
are activated during MES cataptosis.
In summary, secondary palate shelf fusion process can be described as
follows (Fig. 8). Initially,
shelves approach each other until contact is made probably between filopodia
from periderm cells and with the help of proteoglycans. More intimate MEE
contact proceeds through a process that is accompanied by the migration of
periderm cells to the oral and nasal ends. Progressive adhesion appears to be
controlled by periderm cell migration. If this periderm cell migration is not
in place, a thinner palate would form. Periderm cells in the epithelial
triangles could also be important for sealing the ends of the MES. Chimaeric
palates CD1EGFP clearly reveal the intercalation between MEE cells from
each shelf as recently reported (Tudela et
al., 2002
). This process, which results in a single epithelial
sheath, represent a classical convergent extension phenomenon
(Wallingford et al., 2002
),
and cause MES growth in both anteroposterior and oronasal axes. Strong
adhesion between shelves probably originates from this MEE cell intercalation,
and we propose that it is up to this stage that cell death is activated.
Finally, dying cells actively promote the activation of MMPs such as MT1-MMP
and MMP13 that cause basal lamina degradation. Basal lamina initially breaks
down in fragments generating the epithelial pearls that later degenerate to
produce the fused palate.
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ACKNOWLEDGMENTS |
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