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First published online 3 August 2006
doi: 10.1242/dev.02520
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Department of Molecular, Cellular and Craniofacial Biology and Birth Defects Center, University of Louisville, Louisville, KY 40202, USA.
* Author for correspondence (e-mail: j0ding03{at}gwise.louisville.edu)
Accepted 27 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C57BL/6] chimeric culture
system, in which a Rosa26-originated `blue' palatal shelf was paired
with a C57BL/6-derived `white' palatal shelf. Using this organ culture system,
we observed the migration of medial edge epithelial cells to the nasal side,
but not to the oral side. We also observed an anteroposterior migration of
medial edge epithelial cells, which may play an important role in posterior
palate fusion. To examine epithelial-mesenchymal transdifferentiation during
palate fusion, we bred a cytokeratin 14-Cre transgenic line into the
R26R background. In situ hybridization showed that the Cre
transgene is expressed exclusively in the epithelium. However,
ß-galactosidase staining gave extensive signals in the palatal
mesenchymal region during and after palate fusion, demonstrating the
occurrence of an epithelial-mesenchymal transdifferentiation mechanism during
palate fusion. Finally, we showed that Apaf1 mutant mouse embryos are
able to complete palate fusion without DNA fragmentation-mediated programmed
cell death, indicating that this is not essential for palate fusion in
vivo.
Key words: Mouse secondary palate, Cell migration, Apoptosis, Apaf1 mutant mice, K14-Cre transgenic mice, Epithelial-mesenchymal transdifferentiation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Murray and Schutte, 2004).
From E12.5, the two palate shelves grow down vertically along the two sides of
the tongue. Each palate shelf consists of a neural crest-derived mesenchymal
core surrounded by epithelial cells that originate from the cranial ectoderm.
The vertical growth of the palate shelves continues until E13.5
(Ferguson, 1988;
Murray and Schutte, 2004).
At E14.5, the vertical palate shelves elevate above the tongue, grow
horizontally towards each other, and contact each other at the medial edge
epithelium (MEE) region along the facial midline. The contact initiates
adhesion between the two MEE layers, and triggers a series of cellular and
biochemical reactions that lead to the fusion of the palate shelves at E15.5
(Ferguson, 1988;
Murray and Schutte, 2004).
During fusion, the MEE layers from the two palate shelves first merge to form
the MEE seam, which subsequently undergoes degeneration, giving rise to the
formation of a continuous palate that separates the oral and nasal cavities
(Ferguson, 1988).
The cellular mechanism underlying seam degeneration and the fate of the MEE
seam cells have been a major focus of the field for more than a decade.
However, controversies still remain on this issue. Three major models have
been proposed for seam degeneration: epithelial-mesenchymal
transdifferentiation (EMT) (Griffith and
Hay, 1992; Nawshad et al.,
2004; Shuler et al.,
1992); MEE cell apoptosis (programmed cell death)
(Cuervo and Covarrubias, 2004);
and lateral migration of MEE cells (Carette
and Ferguson, 1992).
The cell migration model was first proposed by Carette and Ferguson, who
suggested that the MEE cells may migrate to the oral and nasal sides of the
palatal shelf during fusion (Carette and
Ferguson, 1992). To date, there have only been very limited
studies to evaluate this model experimentally
(Cuervo and Covarrubias,
2004).
The apoptosis model has long been proposed for MEE seam degeneration.
However, only recently have relevant experimental data been obtained regarding
this hypothesis. Cuervo and colleagues examined apoptosis in the MEE seam
during palatal fusion in vivo and in vitro using TdT-mediated dUTP nick end
labeling (TUNEL) assay (Cuervo et al.,
2002; Cuervo and Covarrubias,
2004). Activation of caspase 3 has been detected in the triangular
area of the palate at E15.5 (Vaziri et
al., 2005). As to the functional requirement of apoptosis in
mediating seam degeneration, contradictory results were reported by different
investigators. Cuervo and Covarrubias reported that the addition of cell death
inhibitors, e.g. z-VAD, in palate organ culture prevented seam and basement
membrane degeneration in vitro (Cuervo and
Covarrubias, 2004). By contrast, Takahara and colleagues reported
that palatal shelves treated with caspase inhibitors, YVAD-CHO and DEVD-CHO,
still underwent normal fusion in vitro
(Takahara et al., 2004). It is
important to note that both reports are based on in vitro culture experiments
involving the use of chemical inhibitors. Because cell death is highly
sensitive to the environment and the condition of the cell, one has to be
cautious in applying in vitro cell death data to the in vivo situation. In
addition, specificity is often a concern with the use of chemical
inhibitors.
In contrast to cell migration and cell death, epithelial-mesenchymal
transdifferentiation has been extensively studied. However, the experimental
data on this issue remains inconsistent and controversial. Carette and
Ferguson labeled the mouse palate epithelial cells with Dil, traced the
labeled cells during palatal fusion in vitro, and found no labeled cells in
the mesenchymal region after fusion
(Carette and Ferguson, 1992).
Similar in vitro results were recently obtained by Cuervo and Covarrubias,
using CCFSE as a labeling reagent (Cuervo
and Covarrubias, 2004). By contrast, Schulle and colleagues
approached this question in mouse embryos in vivo by intraperitoneal (IP)
injection of Dil, and observed labeled cells in the mouse palatal mesenchymal
area at E15.5 (Shuler et al.,
1992). Sun and colleagues labeled the chicken palatal epithelium
with CCFSE, cultured the palate pairs in vitro with Tgfß3, and found that
the treated chicken palatal pairs underwent fusion, with a high level of CCFSE
signal present in the medial edge mesenchymal area
(Sun et al., 1998).
Along with the physical labeling approach, a molecular biology based
labeling approach has also been applied to address this issue.
Martinez-Alvarez and colleagues infected the palate shelves with a retroviral
vector that constitutively expressed the bacterial lacZ gene, and
cultured the infected palatal pairs in vitro in complete medium with serum.
Following in vitro palate fusion and ß-galactosidase (ß-gal)
staining, the investigators observed blue cells in the mesenchymal region
(Martinez-Alvarez et al.,
2000). By contrast, Cuervo and Covarrubias infected the palatal
shelves with a lacZ-expressing adenoviral vector and cultured them in
serum-free medium, but no ß-gal-positive cells were observed in the
mesenchymal region after in vitro palatal fusion
(Cuervo and Covarrubias, 2004).
Very recently, a Cre-Loxp-based genetic labeling system has also been
applied to study this issue. Vaziri and colleagues examined mice expressing
Cre recombinase under the control of Shh and cytokeratin 14
regulatory elements in the R26R background, and observed no
ß-gal-positive cells in the mesenchymal region after palatal fusion in
vivo (Vaziri et al., 2005).
However, the labeling intensity in MEE cells prior to fusion was rather low in
that study.
As to the molecular mechanism mediating MEE seam degeneration, Tgfß3
has been shown to be essential for MEE degeneration by a genetic inactivation
approach (Kaartinen et al.,
1995; Proetzel et al.,
1995). Tgfß3 is a growth factor with multiple biological
functions and activities (Massague,
1998). Several mechanisms have been proposed for
Tgfß3-mediated seam degeneration, including facilitating
epithelial-mesenchymal transdifferentiation
(Kaartinen et al., 1997;
Nawshad and Hay, 2003;
Sun et al., 1998), initiating
cell adhesion (Gato et al.,
2002; Sun et al.,
1998), and promoting basement membrane and cell matrix
degeneration (Blavier et al.,
2001).
In this study, we first employed an in vitro [Rosa26C57BL/6]
chimeric culture system to examine epithelial cell migration in cultured
palate fusion in vitro. We also used a Cre-Loxp-based genetic
labeling system, a similar approach to Vaziri and colleagues
(Vaziri et al., 2005), to
label and follow the palate epithelium during palate fusion in vivo. We bred a
cytokeratin 14-Cre (K14-Cre) transgenic mouse line
(Vasioukhin et al., 2001) into
the R26R background (Soriano,
1999). In this combination, the expression of Cre recombinase is
driven by a cytokeratin 14 (K14) promoter and enhancer, a
well-characterized, epithelium-specific regulatory element
(Vasioukhin et al., 2001).
Thus, the R26R reporter locus will be specifically activated and
irreversibly labeled in the epithelium. Finally, to find out whether apoptosis
is functionally required for seam degeneration in vivo, we examined MEE cell
apoptosis and palatal fusion in Apaf-1 deficient mice, in which
caspase 3, a key effector caspase, is not activated in embryonic cells
(Honarpour et al., 2000).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Palatal organ culture
Palatal organ culture was carried out following the procedures described by
Carette and Ferguson (Carette and Ferguson,
1992). Briefly, mouse palatal shelves at E13.5 were dissected out
in cold 
-MEM medium containing 25 mM Hepes. The dissected palate
shelves were paired on MF-Millipore membranes (Fisher AABP 04700) pre-treated
with boiling water followed by 70% ethanol washing. The paired palatal shelves
were then placed on an autoclaved grille in an organ culture dish (Fisher
08-772-12) containing BGJb medium (Invitrogen/Gibco) with 0.1 µg/ml
ascorbic acid, and cultured at 37°C with 5% CO2 for 12, 24, 48
or 60 hours with a medium change every 24 hours.
ß-galactosidase (ß-gal) staining, in situ hybridization and TUNEL assay
Whole-mount ß-galactosidase staining was carried out according to the
standard protocol described by Nagy et al.
(Nagy et al., 2003). For
[Rosa26C57BL/6] chimeric culture, the ß-gal-stained
explants were processed for OCT embedding, and 12 µm cryosections were cut.
A total number of 25 cultures have been examined in this study. For the
[K14-Cre; R26R] experiment, the [K14-Cre;
R26R] embryonic heads were identified by positive ß-gal staining
without genotyping. The blue embryonic heads were then processed for paraffin
embedding and 10 µm paraffin sections were cut. In this study, we have
analyzed 18 blue embryos at E14.5 and 16 blue embryos at E15.5. Both
cryosections and paraffin sections were counter stained with Nuclear Fast Red
(Vector Laboratories).
In situ hybridization of the Cre gene with E13.5, E14.5 and E15.5
K14-Cre positive heads was carried out according to Shen
(Shen, 2001). For each stage,
at least three samples were included.
TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed using an In Situ Cell Death Detection Kit (Roche), following the manufacturer's instructions. The TUNEL assay was carried out on fixed paraffin-embedded tissues, which were then mounted without counter staining. Three wild-type palates and four Apaf1-/- palates were assayed in this study.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C57BL/6] chimeric culture in which a palatal
shelf dissected from Rosa26 mice
(Zambrowicz et al., 1997) was
paired with another palatal shelf dissected from C57BL/6 mice. We assumed
that, if lateral migration of MEE seam cells occurs during palatal fusion in
vitro, the cells that emerge from the seam will split randomly to either side
of the pair, and that the Rosa26-originated epithelial cells may be
present in the oral or nasal side of the C57BL/6 half of the pair and can be
detected by ß-gal staining. In other words, if ß-gal-positive
epithelial cells can be detected in the C57BL/6 palate, it is reasonable to
believe that the lateral migration of MEE seam cells occurs during palatal
fusion, at least in vitro. As shown in Fig.
1B, ß-gal-positive cells are indeed present in the C57BL/6
palatal nasal epithelium in a [Rosa26C57BL/6] chimeric pair
cultured oral side up, indicating a migration of cells to the nasal side of
the palate during fusion in vitro (Fig.
1B). Notably, all the [Rosa26C57BL/6] chimeric
explants examined so far (n>15) have shown emerging cells on only
the nasal side of the C57BL/6 palate, not on the oral side. To exclude the
possibility of a positional effect, we cultured the palate explants nasal side
up; however, we still found ß-gal-positive cells present only in the
nasal sides of the C57BL/6 palates (data not shown). We therefore conclude
that MEE seam cells can migrate, at least to the nasal side during in vitro
palatal fusion, whereas migration to the oral side is still an unsolved
question. Interestingly, we observed ß-gal-positive cells in the C57BL/6
palate in posterior regions in which the two shelves are still separated
(Fig. 1C). This observation
prompted us to hypothesize that the MEE seam cells may also migrate along the
anteroposterior (AP) axis. To confirm this possibility, we sectioned the
paired explants in the manner indicated in
Fig. 1D. We found that
ß-gal-positive cells did indeed extend posteriorly
(Fig. 1E). Because the blue
staining in the C57BL/6 palate is continuous rather than dispersed
(Fig. 1C,E), it is unlikely
that the blue signals in the C57BL/6 side are the cell attachments from the
Rosa26 palate. Based on these observations, it is likely that during
in vitro palatal fusion, MEE cells commit to migration in two directions:
migrating nasally along the oral-nasal axis; and migrating posteriorly along
the AP axis (see Fig. 6 for a
better illustration). The AP migration may play a crucial role in posterior
palate fusion by `zipping' the posterior palate together, as it has been
suggested that the fusion in the posterior region is different from that in
the middle regions (see Discussion for details).
|
) in
the R26R background (Soriano,
1999) to generate [K14-Cre; R26R] embryos.
Because the Cre transgene is under the control of an
epithelial-specific K14 regulatory element
(Vasioukhin et al., 2001), the
R26R reporter locus will be activated only in epithelial cells and
will lead to irreversible ß-gal labeling of those cells. Therefore, the
ß-gal-positive cells in [K14-Cre; R26R] embryos are
epithelial in origin, and the presence of ß-gal-positive cells in palatal
mesenchymal region after fusion will be an indication of
epithelial-mesenchymal transdifferentiation. We first carried out in situ hybridization with the Cre anti-sense RNA probe in [K14-Cre; R26R] embryos to validate that the expression of the Cre transgene in those embryos is indeed epithelium specific. We found that the Cre transgene is highly expressed in the entire palatal epithelium at E13.5 (Fig. 2B). At E14.5, the expression becomes heterogeneous along the AP axis of palate. In the anterior and posterior region, the Cre transgene is expressed roughly in the entire palate epithelium (Fig. 2C,E), whereas expression in the middle region is restricted to the oral side of palate epithelium and the triangular area (Fig. 2D). The expression of the Cre transgene becomes undetectable in the palate at E15.5 (data not shown). No expression was observed in mesenchymal cells throughout palate development. Therefore, the Cre transgene in this K14-Cre line confers an epithelium-specific expression, at least in the development of the secondary palate.
We then examined the ß-gal staining pattern in palate development in the [K14-Cre; R26R] embryos. We found that the ß-gal labeling intensity in the palatal epithelium varies among the [K14-Cre; R26R] embryos, ranging from high density to almost no labeling (data not shown). Moreover, the labeling efficiency also varies with respect to the region of palatal epithelium, as some [K14-Cre; R26R] embryos are strongly labeled in the oral and nasal palatal epithelium, but are weakly labeled in the MEE seam, whereas others may be strongly labeled in the MEE seam region, but poorly labeled in oral and nasal sides of palate. This phenomenon is frequently observed in various Cre mice. Therefore, the labeling level in the oral or nasal palatal epithelium does not necessarily reflect the labeling situation in the seam. In order to trace MEE fate during fusion, it is essential that the [K14-Cre; R26R] embryos selected for the analysis are strongly labeled with ß-gal in their MEE seams. For that purpose, we focused on the stages between E14.5 and early E15.5, in which the seam had not completely vanished and the remaining seam can be used as an internal control for the ß-gal-labeling situation in the seam cells. Fig. 3A shows the ß-gal staining in the anterior palate of a [K14-Cre; R26R] embryo at early E14.5, in which the two palate shelves had just made contact. The MEE region of this embryo is strongly labeled with ß-gal, but no ß-gal signal was detected in the mesenchymal cells, particularly the medial edge mesenchyme region, indicating that no EMT had occurred before fusion (Fig. 3A). In the initial stage of seam degeneration, some ß-gal-positive cells are located off the midline within the mesenchymal region (Fig. 3B). Around late E14.5 and E15.0, seam degeneration is more advanced and more ß-gal-positive cells were found away from the midline; in addition, a large portion of medial edge mesenchymal cells are ß-gal positive (Fig. 3C,D), indicating that epithelial-mesenchymal transdifferentiation occurs during seam degeneration. At E15.5, the seam degeneration is complete, and a large patch of ß-gal-positive cells with typical mesenchymal cell morphology is present in the medial edge region (Fig. 3E). The high intensity of ß-gal staining in the degenerating seam indicated that the MEE seams of the examined embryos are strongly labeled with ß-gal (Fig. 3B-D). In this experiment, eight out of 18 embryos at E14.5, and eight out of 16 embryos at E15.5, gave high-level labeling in the degenerating or residual seam cells, and all of them showed good signals in the mesenchymal region. These results demonstrate that EMT does occur during palate fusion in vivo.
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|
;
Cuervo and Covarrubias, 2004),
and a recent study suggested that the apoptosis in palatal MEE cells is
mediated by caspase 3 (Vaziri et al.,
2005). To determine whether apoptosis is essential for palate
fusion and seam degeneration during mouse development in vivo, we examined
palate fusion in Apaf1 mutant mouse embryos. The mouse Apaf1
gene is the mouse homolog of the C. elegans CED-4 gene, which encodes
a crucial component in the caspase-3-mediated apoptosis pathway
(Green and Reed, 1998).
Apaf1 mutant mouse embryos are deficient in caspase 3 activation and
display enormous apoptotic defects during embryonic development
(Green and Reed, 1998;
Honarpour et al., 2000).
Surprisingly, we found that complete palate fusion still occurs in the
Apaf1 mutant embryos. Histological analysis showed that the
Apaf1 mutant palate forms a normal MEE seam at E14.5
(Fig. 4D), which undergoes
degeneration at E15.5 (n=14; Fig.
4E). At E16.5 (n=10), the mutant embryos form a
continuous palate with no residual seam cells
(Fig. 4F). Interestingly, the
mutant palates at E15.5 often present an enlarged triangular area that
presents with more compacted cell density than wild-type palates
(Fig. 5A,B). To rule out the
possibility that caspase 3-independent apoptosis occurs in the Apaf1
mutant palate, we carried out the TUNEL assay in both wild-type and
Apaf1 mutant embryos. We found few TUNEL-positive cells in the palate
epithelium prior to E15.5 in wild-type embryos (data not shown), and apoptotic
palate MEE cells become detectable as late as E15.5
(Fig. 5C). The majority of
apoptotic cells are in the triangular area
(Fig. 5C). This data is
consistent with the previously reported capase 3 activation in the triangular
area of the palate at E15.5 (Vaziri et
al., 2005). The TUNEL-positive cells are completely abolished in
the Apaf1 mutant palate at E15.5
(Fig. 5D). Therefore, the
Apaf1 mutant palate cells do not commit to DNA fragmentation-mediated
programmed cell death. On the basis of these results, we conclude that
apoptosis is not essential for palate fusion in vivo. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C57BL/6] chimeric palate
pairs uncovered a three-dimensional movement of MEE seam cells during palate
fusion: lateral migration to the nasal side of the palate and AP migration
from the middle to the posterior. Interestingly, lateral migration to the oral
side of the palate was not observed in this analysis. It is not clear whether
lateral migration to the oral side of palate is blocked by an intrinsic
mechanism or whether the lack of migration is simply due to the in vitro
culture condition. Cuervo and Covarrubias reported lateral migration to both
the nasal and oral side of the palate in culture using an eGFP-labeling system
(Cuervo and Covarrubias, 2004).
However, the cellular resolution was rather low in that reported study.
|
),
whereas the AP migration has not been reported previously. We speculate here
that the AP migration may serve the dual functions of promoting seam
degeneration and facilitating posterior palate fusion. Owing to palate
topology, the two palate shelves at E14.5 make close contact only in the
middle region, whereas the posterior regions are widely separated, as
illustrated in Fig. 2A. How the
posterior palatal shelves achieve tight contact at E15.5 is still an
unanswered question in palate development. The AP migration of MEE seam cells
may serve as a `zipper' to bring the two parts together.
|
Epithelial-mesenchymal transdifferentiation during palate fusion in vivo
Our ß-gal staining with [K14-Cre; R26R] embryos revealed that
a significant number of mesenchymal cells are ß-gal positive during and
after palate fusion. Because our in situ data showed that the expression of
the Cre transgene in the palate is exclusively restricted to the
palatal epithelium, the ß-gal-positive mesenchymal cells are epithelial
in origin. We therefore provide strong evidence to support the occurrence of
the long debated epithelial-mesenchymal transdifferentiation during palate
fusion. Our results are different from a recent report by Vaziri and
colleagues (Vaziri et al.,
2005), who used a similar approach with a different
K14-Cre transgenic line. The discrepancy may reside in the different
Cre transgenic lines used in the studies. The ß-gal-labeling
level in MEE prior to fusion was rather low in that reported study, which
could have meant that the signal was not sufficient to be detected. In
addition, the embryos selected for analysis in that study demonstrated good
palate fusion with almost no residual seam left. Because the labeling level in
the oral and nasal palate epithelium does not necessarily correlate with the
labeling situation in MEE, it is not clear how well the MEE seam cells were
labeled in the embryos analyzed in the reported study.
|
),
whereas another study reported that palate fusion occurs normally in vitro in
the presence of the caspase inhibitors YVAD-CHO and DEVD-CHO
(Takahara et al., 2004). In
our current study, we took a genetic approach to address this issue in vivo.
The mouse Apaf1 gene is the mouse homolog of the C. elegans
CED-4 gene that encodes a protein factor essential for capase activation
(Green and Reed, 1998).
Targeted disruption of Apaf1 in mice leads to the failure of caspase
3 activation and numerous cell death-related defects
(Cecconi et al., 1998;
Honarpour et al., 2000). In
the present study, we found that DNA fragmentation-mediated apoptosis was
completely abolished in the palates of Apaf1 mutant embryos, as
judged by the TUNEL assay (Fig.
5D). However, palate fusion and seam degeneration did occur in the
Apaf1 mutant embryo (Fig.
4D-F). It is worth mentioning that a previous study with a
different line of Apaf1 mutant mice described the persistence of the
palate seam in the absence of the Apaf1 gene
(Cecconi et al., 1998).
However, the analysis in that study was only until E14.5, at which time the
seam in a wild-type embryo is still evident. Cuervo and Covarrubias reported a
much higher number of apoptotic cells in the MEE seam of in vitro-cultured
palate pairs (Cuervo and Covarrubias,
2004). As apoptosis is very sensitive to the environment and the
condition of the cells, the increased apoptotic cells in the seam of the
cultured palate may have been due to some kind of artificial increase in an in
vitro system. Our analysis with Apaf1 mutant embryos strongly demonstrates that the function of the observed apoptosis is not essential for palate fusion and seam degeneration. However, this does not mean that apoptosis does not contribute to palate fusion and seam degeneration during normal palate development, as it is obvious that MEE cell death can at least accelerate the removal of the MEE seam during palate fusion. In fact, Apaf1 mutant palates at E15.5 display abnormal-looking triangular areas that are larger and more compacted (Fig. 5B). This suggests that programmed cell death may play a role in the clearance of the triangle cells. It is also possible that more MEE cells commit to cell migration to the triangular areas when the cell death pathway is impaired.
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ACKNOWLEDGMENTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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