1 Dulbecco Telethon Institute, Department of Biology, University of Rome `Tor
Vergata', via della Ricerca Scientifica, 00133 Rome, Italy
2 Department of Pathology, University of Alabama at Birmingham, SC 961E, 1530
Third Avenue South, Birmingham, AL 35294, USA
3 P. K. Anokhin Institute of Normal Physiology RAMS, 6, Bol. Nikitskaya st,
103009 Moscow, Russia
4 Department of Molecular Cell Biology, Max-Planck Institute of Biophysical
Chemistry, Am Fassberg 11, 37077 Goettingen, Germany
5 Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00710
Helsinki, Finland
* Author for correspondence (e-mail: marjo.salminen{at}helsinki.fi)
Accepted 19 January 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Apoptosis, Bcl2l, Bcl-XL, Bcl-XS, Caspase 9, Proliferation, Otic vesicle closure, Semicircular ducts, Cochlea, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mammalian inner ear includes specialized vestibular sensory organs for
balance and a coiled cochlea for hearing. The early development of the inner
ear involves invagination of the ectodermal placode and its closure to form
the otic vesicle. Soon after closure, the otic epithelium undergoes a sequence
of complex morphogenetic events to form the membranous labyrinth. The
endolymphatic duct extends dorsally and the cochlear duct grows ventrally. The
utricle and saccule are separated from each other and from the cochlear duct
through deepening constrictions in the ventral vestibular part, whereas the
dorsal portion develops into three semicircular ducts through a multi-step
process. These ducts first emerge as bilayered epithelial outpocketings. The
two opposing layers then approach each other and form a so-called fusion
plate, in which the cells first detach from the underlying basement membrane,
intercalate and then disappear, creating a hollow duct which further grows out
to obtain its adult form and size (Sher,
1971; Martin and Swanson,
1993
). The periotic mesenchyme develops in close contact with the
otic epithelium and forms the bony otic capsule to surround the membranous
labyrinth (Sher, 1971
;
Martin and Swanson, 1993
).
Apoptotic cells have been detected in restricted areas of the developing
vertebrate inner ear. These areas include the epithelial cells that
transiently connect the surface ectoderm and the otic vesicle during and after
its closure, the base of the outgrowing endolymphatic duct, a ventral area
above the primordium of the vestibulocochlear ganglion and the ventro-medial
wall of the growing cochlear duct
(Marovitz et al., 1976;
Marovitz et al., 1977
;
Represa et al., 1990
;
Fekete et al., 1997
; Nikolic
et al., 2000). In chicken embryos, apoptosis has also been detected in the
fusion plates of the semicircular ducts
(Fekete et al., 1997
).
Apoptosis at the fusion plates has not been detected in mouse or human embryos
(Martin and Swanson, 1993
;
Nishikori et al., 1999
).
Although the anatomic distribution of apoptotic cells in the inner ear has
been described, the molecular pathways regulating programmed cell death in
this structure have not been studied in detail. The Apaf1 mutant mice
die usually perinatally (Cecconi et al.,
1998; Yoshida et al.,
1998
). A few postnatal survivors show a hyperactive behavior that
was suspected to be a consequence of inner ear defects, because the brain of
these mice appeared completely normal
(Honarpour et al., 2000
).
Postnatal defects have been reported in the inner ear of caspase 3 mutant mice
(Takahashi et al., 2001
). Here
we undertook an analysis of Apaf1, Bcl2l and caspase 9 mutant mice in
order to gain insight into the molecular mechanisms controlling cell death
during inner ear morphogenesis. We observed that the majority of the cell
death in the inner ear was Apaf1-dependent and most probably occurred through
the Apaf1/caspase 9 apoptosome pathway. The lack of apoptosis in
Apaf1 mutant mouse embryos led to widespread defects in epithelial
morphogenesis and outgrowth. In contrast, increased apoptosis in
Bcl2l mutant embryos had no major impact on morphogenesis. However,
developmental defects with the latter mutants co-localized with a decrease of
apoptosis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histological analysis and immunohistochemistry
For histological analysis serial paraffin sections through the inner ear
were stained with Hematoxylin and Eosin or Giemsa. Immunohistochemical
detection of activated caspase 3 was performed with the rabbit anti-CM1
antibody as before (Cecconi et al.,
1998). For Bcl-XL immunostaining with mouse
anti-Bcl-XL (H-5) antibody (Santa Cruz), Bouin's-fixed paraffin
sections were deparafinized and boiled for 20 minutes in 10 mM Citrate buffer
(pH 6). After secondary antibody (donkey anti-mouse HRP IgG) treatment, the
signal was amplified using the TSA Cyanine 3 System Kit (PerkinElmer Life
Science Products). BrdU intraperitoneal injection and detection in
proliferating cells were performed as before
(Salminen et al., 2000
).
Anti-class III ß-tubulin (TUJ1, Babco) was used as a control antibody.
Three-dimensional reconstructions from histological sections of inner ears at
stage E13.5 were constituted from serial paraffin cross-sections of 10 µm
thickness. One ear from each genotype was used for reconstruction and the
embryos all came from the same litter of a mating between two
Apaf1/Bcl2l double heterozygous animals.
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) assay
Total RNA was isolated with Trizol Reagent (Life Technologies) from
wild-type E9.5 and E11.5 mouse embryos. To detect the different splice
variants produced from the Bcl2l gene, 2 µg of the isolated RNA
was subjected to RT reaction using the RT-primer
5'-GACTGAAGAGTGAGCCCAGATC-3'. The forward and reverse primers for
PCR were 5'-CAGTGAAGCAAGCGCTGAGAG-3' and
5'-CGTCAGAAACCAGCGGTTGAAG-3', respectively.
RNA in situ hybridization and staining for ß-galactosidase activity
In situ hybridization and preparation of the radioactive antisense and
sense Netrin1 (Serafini et al.,
1996), Pax2 (Dressler
et al., 1990
) and Dlx5
(Acampora et al., 1999
) probes
were performed as described in Salminen et al.
(Salminen et al., 2000
).
Whole-mount lacZ staining for E12.5
Apaf1+/ and -/- embryos was conducted as
described in Hogan et al. (Hogan et al.,
1994
).
Detection of apoptosis
Detection of apoptosis was performed with the in situ DNA-end labeling
technique, the TUNEL method, that detects DNA fragmentation as an indicator of
ongoing apoptosis. TUNEL analysis was performed on 10 µm-thick paraffin
sections with the ApopTag Fluorescein In Situ Apoptosis Detection Kit
(Intergen). The proportion of TUNEL-positive cells from all otic vesicle cells
was calculated on serial sections of 2 or 3 E9.5, E10.5 or E12.5 embryos.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Bcl-XL immunoreactivity was found all over the otic epithelium at E10.5-12.5, but no protein could be detected in the periotic mesenchyme (Fig. 1G-I). To analyze the expression of the alternatively spliced Bcl-XS isoform we performed RT-PCR reactions on total RNA isolated from E9.5 and E11.5 otic vesicle and periotic mesenchyme. The RT-PCR primers were designed to hybridize to sequences common to both Bcl-XL and Bcl-XS so that Bcl-XL-specific mRNA would give a 395 bp-long fragment and Bcl-XS-specific mRNA a 206 bp-long fragment. As seen in Fig. 1N, only the Bcl-XL-specific mRNA could be detected at E9.5 and E11.5.
Changes in apoptotic profiles at E9.5-10.5 in the Apaf1 and Bcl2l mutant inner ears
We compared the pattern of programmed cell death in Apaf1 and
Bcl2l mutant inner ears with the corresponding wild-type littermates.
In an early otic vesicle, at E9.5-10.0, a TUNEL-positive cell cluster
localized to the ventro-posterior area (arrowhead
Fig. 2B) and a few scattered
dying cells could be observed more dorsally. In Apaf1 mutant otic
vesicles, no apoptotic cells were detected at E9.5-10.5
(Fig. 2C and data not shown).
In Bcl2l mutant otic epithelium, a significant 3-fold increase in the
proportion of TUNEL-positive cells could be observed
(Fig. 2D,
Table 1).
|
|
A marked 3-fold increase in the ratio of TUNEL-positive cells could be
detected in the endolymphatic duct epithelium of the Bcl2l mutant
embryos at E10.5 (Table 1). In
addition, the distribution of dying cells in Bcl2l mutant ears
extended more ventrally (Fig.
2H). In the rest of the otic vesicle, a 2-fold increase of
TUNEL-positive cells could be observed
(Table 1). A cluster of
apoptotic cells could be observed in the ventro-posterior area of the
Bcl2l mutant otic epithelium, but the broad area of apoptotic cells
observed more dorsally in the wild-type otic vesicle
(Fig. 2G) was not detected
(Fig. 2H). Extensive cell
movement occurs in the otic epithelium after its closure
(Brigande et al., 2000) and
thus, the lack of dying cells in the dorsal wall of the Bcl2l mutant
otic vesicle could be because of premature death of these cells while still in
a more ventro-posterior location (Fig.
2B,D).
Apoptosis has been thought to be responsible for the complete detachment of
the mammalian otic vesicle from the surface ectoderm by removing the
connecting epithelial stalk after the closure
(Marovitz et al., 1976;
Represa et al., 1990
;
Lang et al., 2000
). We
observed a cluster of apoptotic cells in the region between the surface
ectoderm and the endolymphatic duct epithelium in wild-type embryos at E9.5
and E10.5 (Fig. 2F and data not
shown). In the Apaf1 mutant embryos (n=3), this apoptotic
group of cells could not be observed but the otic vesicles had nevertheless
closed normally (Fig. 2C). In
Bcl2l mutant embryos (n=3) no clear cluster of apoptotic
cells but instead a few scattered cells could be observed in the closure
region (Fig. 2H).
Very little or no apoptosis could be observed in the periotic mesenchyme of the wild-type embryos (Fig. 2F,G). Apoptosis could, however, be detected elsewhere in the head mesenchyme (Fig. 2B,F). Also, no apoptosis could be observed in the periotic mesenchyme of either Apaf1 or Bcl2l mutant embryos (Fig. 2C,D,H).
Taken together, our results demonstrate that the normal developmental apoptotic pattern had changed in both Apaf1 and in Bcl2l mutant inner ears. The almost complete lack of apoptotic cells in the Apaf1 mutant ears indicates that the majority of the apoptosis detected in the wild-type otic epithelium at E9.5-10.5 is Apaf1-dependent. Inactivation of the Bcl2l gene led to a general increase of cell death in the expected apoptotic areas of the otic epithelium. This result is probably because of the lack of the anti-apoptotic Bcl2l isoform. However, at the closure site, a decrease of apoptotic cell numbers was observed, suggesting that pro-apototic Bcl2l isoforms might be active there.
Changes in the apoptotic pattern at E12.5
In wild-type embryos at E12.5, TUNEL-positive cells were detected in the
endolymphatic duct epithelium (Fig.
3A), the wall of the cochlear duct
(Fig. 3A-D), the distal edge of
the three semicircular duct epithelium (shown for the superior duct in
Fig. 3A-D, arrow in A) and the
areas where the utricle, saccule and the cochlear duct will be separated by
deepening constrictions (Fig.
3B, arrowheads). In addition, some apoptotic cells were observed
in the superior and posterior semicircular duct epithelium prior to the fusion
in the area that is destined to form the fusion plate. A few dying cells were
also detected in the immediately adjacent periotic mesenchyme
(Fig. 3A,G, arrowheads). In
accordance with previous observations
(Martin and Swanson, 1993), we
could not detect any dying cells when the two epithelial cell layers were
opposed and fused (data not shown). However, a few TUNEL-positive cells were
observed in the mesenchyme lining the small empty hole after the fusion plate
had been cleared (arrowheads in Fig.
3C,D). In general, no or very little apoptosis occurred in other
areas of the periotic mesenchyme at this stage.
|
In summary, our results suggest that very little or no Apaf1-independent apoptosis occurs in the otic epithelium at E12.5. In the Bcl2l mutant embryos a general increase of apoptosis was observed as at earlier stages, except for the fusion plate area of the posterior semicircular duct where a lack of apoptotic cells was observed. Very little apoptosis can be detected in the periotic mesenchyme, suggesting that apoptosis is not critical for the formation of the otic capsule.
Three-dimensional reconstructions of the mutant inner ears reveal specific morphogenetic defects and reduced growth
The morphogenetic architecture of the membranous labyrinth has been
established by E13.5. To examine the morphology of the epithelial labyrinths
of the embryos at this stage we reconstructed three-dimensional images from
serial sections. The reconstructions showed that both Apaf1 and
Bcl2l mutant ears were smaller than the wild-type ears and were
reduced to 54% and 70%, respectively (Fig.
4A-C). The diameter of the corresponding mutant heads were 86% and
77% of that of the wild-type heads, respectively (data not shown), which could
explain the reduction in membranous labyrinth size in the case of
Bcl2l mutants but not in the case of Apaf1 mutants.
|
|
The three-dimensional reconstructions demonstrated that the decrease of apoptosis in Apaf1 mutant embryos led to a growth retardation and to defects in shaping or `sculpting' the membranous labyrinth. Bcl2l inactivation led only to a restricted phenotype concerning the posterior semicircular duct.
Superior semicircular duct formation is most severely affected in Apaf1 mutant embryos
Some variation in the severity of the phenotype of the Apaf1
mutant ears could be observed when sections through the ears of 16 E12.5-18.5
embryos were analyzed. General variability in Apaf1 mutant phenotypes
has been observed before. In the case of brain, variation ranges from severely
overgrown nervous tissue to a completely normal-looking tissue
(Cecconi et al., 1998;
Honarpour et al., 2000
). We
obtained embryos at a ratio of 10/6 for severe/mild phenotypes judged
according to the brain overgrowth. The severity of the inner ear morphological
phenotype seemed to correlate to some extent with that in the brain. The
Apaf1 mutant inner ear in Fig.
4B,E comes from an embryo with a highly overgrown and open brain
structure, whereas Fig. 5B,C
shows the two ears from an embryo with no obvious brain defects. In embryos in
which brain overgrowth was extensive, the ear was often aberrantly positioned
directly ventrally from the neural tube instead of its normal lateral position
(Fig. 4E). In addition, the ear
had turned in a more horizontal position. However, irrespective of the
severity of the brain phenotype or ear position, the inner ear epithelium was
always reduced except for the endolymphatic duct which was always enlarged
(Fig. 4B,
Fig. 5B,C). In addition, the
superior semicircular duct was always markedly reduced
(Fig. 5B) and sometimes
completely absent (41%) even in embryos with no detectable brain phenotype
(asterisk in Fig. 5C). The
other two semicircular ducts always formed and a clearing of the fusion plates
could be detected by E14.5 (Fig.
4E, Fig. 5B,C).
The periotic mesenchyme seems to develop normally as judged from those Apaf1 mutant embryos that lived until E16.5-18.5 (n=5). The mesenchymal cells closest to the otic epithelium are destined to undergo cell death once the main morphogenetic events have occurred to generate the perilymphatic space between the membranous and bony labyrinths. Interestingly, this late cell death seems to occur normally in the absence of Apaf1 (Fig. 5A-C).
The inner ear phenotype in the caspase 9 mutant embryos closely resembled that of Apaf1 mutants
The results from the analyses of Apaf1 mutant animals emphasized
the importance of Apaf1 for the developmental apoptosis that occurs during
otic epithelium morphogenesis. To determine whether this cell death involves
the apoptosome or occurs through an apoptosome-independent pathway
(Marsden et al., 2002), we
analyzed the inner ear phenotype in six caspase 9 mutant embryos at
E13.5-17.5.
The inner ear phenotype in caspase 9 mutant embryos including morphogenetic
changes (enlarged endolymphatic duct), smaller size, reduced semicircular
ducts and variation in phenotype severity was very similar to that observed in
Apaf1 mutant embryos (Fig.
5D-F and data not shown). As for other organs affected, the
caspase 9 phenotype appeared slightly milder than the Apaf1 phenotype
(Cecconi et al., 1998;
Yoshida et al., 1998
;
Kuida et al., 1998
;
Hakem et al., 1998
). These
results support the idea that most of the cell death in the developing inner
ear occurs through the activation of the apoptosome complex.
Dorso-ventral compartmentalization occurred normally in Apaf1 and Bcl2l mutant otic epithelium
To verify whether the regionalization of the inner ear epithelium occurred
normally in Apaf1 and Bcl2l mutant inner ears, we performed
in situ hybridization analyses with marker genes known to be specific for
either dorsal or ventral parts of the inner ear. The Dlx5 gene was
expressed normally in the dorsal vestibular part and in the endolymphatic duct
of both mutants (shown for wild-type and Apaf1 mutant in
Fig. 6A,B). Pax2
expression was detected ventrally along the utricular wall and in the
developing cochlear duct (shown for wild-type and Bcl2l mutant in
Fig. 6C,D). Netrin1 is
specifically expressed in the semicircular duct fusion-plate-forming
epithelium and after duct formation it remains expressed at the inner edge of
the duct epithelium (Salminen et al.,
2000). In both Apaf1 and Bcl2l mutant ears
Netrin1 was expressed in the fusion plate-forming cells as well as
the newly formed ducts (Fig.
4D-F, Fig. 6E-F).
These results demonstrate that in spite of the changes in apoptotic patterns,
abnormal cell positional identities could not be detected in the mutants.
|
In the severely affected Apaf1 mutant embryos, the overall size of the otic epithelium was clearly diminished already at E11.5 (Fig. 7A,B). In these ears, 22.9%±5.5% of the cells were proliferating in comparison with the wild-type ears in which the proliferation index was 33.4%±4.9% (P<0.001, Student's t-test). At E12.5 the defect in the semicircular duct outgrowth became evident (compare superior and lateral ducts in Fig. 7F,G). However, no clear difference in the proliferation index could no longer be detected here (data not shown). The consequence of a decrease in the proliferation index at an earlier stage could be observed as a strong reduction in the number of cells in the mutant duct area at later stages. At E12.5 the proliferation index in the cochlear duct was 40.4%±10.3% in the Apaf1 mutant and 68.8%±13.6% (P<0.001) in the wild-type (examples in Fig. 7G,H). Moreover, an almost complete lack of proliferating cells was observed in the outer wall of the turning cochlear duct (arrowheads in Fig. 7G,H). No clear change in the local cell proliferation could be observed in the endolymphatic duct epithelium between wild-type and Apaf1 mutant embryos at E11.5 (data not shown).
|
Taken together, our results suggest that Apaf1-dependent apoptosis might be required to achieve the normal level of cell proliferation in the developing otic epithelium excluding the area of the endolymphatic duct. Lack of apoptosis seemed to especially affect the outgrowth of the semicircular and the cochlear ducts. Furthermore, an increase in apoptosis in Bcl2l mutant inner ears seems to result in an increase of proliferation at early stages that could at least partially compensate for the important loss of cells through excess cell death.
Otic vesicle closure failed to occur in Apaf1/Bcl2l double mutant mice
In order to check whether the anti-apoptotic Bcl2l and the pro-apototic
Apaf1 operated independently of each other or through a common pathway during
inner ear development, we generated double mutant embryos. These embryos died
at E12.5-13.0, similar to the Bcl2l single mutants, and suffered from
a hematopoietic failure indicating that the inactivation of Apaf1
cannot inhibit the excessive cell death occurring in the hematopoietic system
when the Bcl2l gene is inactivated
(Motoyama et al., 1995). Apaf1
deficiency did, however, prevent the increased cell death observed in immature
neurons throughout the embryonic Bcl2l-deficient nervous system
(Motoyama et al., 1995
) (data
not shown). The same observations have been reported by Yoshida et al.
(Yoshida et al., 2002
).
We analyzed the inner ear phenotype in three double mutant embryos at E11.5. In none of the three cases did the otic epithelium undergo massive apoptosis as in Bcl2l mutants. Instead, no apoptosis could be detected, a phenotype very similar to that of Apaf1 mutant inner ears (Fig. 8A-B). This result suggests that in contrast to the hematopoietic system, the excess of cell death that results from Bcl2l inactivation requires Apaf1 in the inner ear.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The morphogenesis of the three semicircular ducts is a complex procedure,
the main steps of which seem to be common for all of them. However, targeted
mutations of several genes in mouse have shown selective defects in duct
formation and suggested that the formation of each duct might be independently
controlled by specific sets of genes
(Fekete, 1999). Here we show
that the inactivation of the Bcl2l gene selectively affects the
formation of the posterior semicircular duct. This phenotype could be because
of the observed lack of local apoptosis at the fusion plate-forming epithelium
and/or in the adjacent periotic mesenchyme, suggesting that pro-apoptotic
Bcl2l isoforms may have a developmental role there. Fekete et al.
(Fekete et al., 1997
) have
shown that overexpression of the Bcl2 gene in chicken embryos leads
to a block of cell death that most severely affects the posterior duct. These
observations suggest a special regulatory role for Bcl2 family members
concerning the development of the posterior duct of the two species.
Interestingly, Apaf1 also seems to be required especially for one of the
semicircular ducts; the growth of the superior duct is most severely affected
in Apaf1 mutant embryos.
Some variation of the Apaf1 mutant inner ear development was observed and the severity of the phenotype correlated with that observed in the brain. Therefore, it is possible that a severely overgrown hindbrain enhances the aberrant ear phenotype. However, we observed that the principal structural and growth defects were present also in Apaf1 mutant embryos where no brain phenotype could be detected.
Apoptosis and outgrowth of the inner ear epithelium
Unneeded cells are removed by apoptosis in many organs during development.
The phenomenon is most striking in brain where in some areas nearly 50% of the
cells are eliminated (reviewed by D'Mello,
1998). In Apaf1 mutant mice, there is a decrease in
apoptosis in many neuronal populations including neural precursors. This
results in an increased amount of cells undergoing cell division and to
overgrowth of the brain mass (Cecconi et
al., 1998
; Yoshida et al.,
1998
).
A balance between cell proliferation and apoptosis also seems to play a
critical role during inner ear development. There are regional differences in
the proliferative activity in the otic vesicle resulting in differential
outgrowth of the epithelium (Lang et al.,
2000). In some areas where cells undergo active proliferation,
dying cells are abundant. These areas include the distal edges of the
semicircular ducts and the wall of the cochlear duct
(Fekete et al., 1997
;
Nicolic et al., 2000
). In
Apaf1 mutant embryos, a surprising size reduction of the membranous
labyrinth was already observed at E11.5 and cell proliferation had diminished
by 10-30% depending on the area and stage analyzed. There was an especially
clear reduction in the elongation of the cochlear duct and the outgrowth of
the semicircular ducts. Therefore, the morphogenetic defects observed in the
cochlea and in the semicircular ducts seem to be a consequence of decreased
proliferation together with decreased apoptosis.
How does the lack of apoptosis affect cell proliferation in these
outgrowing areas of the otic epithelium? Apoptosis might stimulate
proliferation through increasing locally the amount of growth-promoting
factors released to the environment. This kind of passive release of
functional factors into the environment from dying cells has been described
(Scaffidi et al., 2002).
Fibroblast growth factors (FGFs) are potential candidates to promote otic
growth because several of them are expressed in the otic epithelium during its
outgrowth period (reviewed by Noramly and
Graigner, 2002
). Interestingly, a subset of extracellular FGFs do
not have any signal peptide and it has been proposed that they could be
released passively from damaged cells (reviewed by
Ornitz and Itoh, 2001
).
In Bcl2l mutant inner ears a dramatic increase of apoptosis was
observed at certain locations at E9.5-10.5. The excess of cell death seems to
be at some extent compensated by an increase in cell proliferation because the
ear is not smaller than in the wild-type littermates at this early stage. In
mice with a targeted mutation in the Bcl2 gene an increase in
apoptosis can be observed during development of many organs. This leads to a
dramatic decrease in the overall size of some organs such as spleen, kidney
and thymus. However, some cell types in the developing kidney, such as the
epithelium and interstitium, hyperproliferated, leading to increased numbers
of these cell types (Veis et al.,
1993). Thus, it seems that in some organs and cell types increased
apoptosis may lead to overproliferation and decreased apoptosis to a growth
delay.
More direct molecular links between proliferation and cell death have been
proposed in several cell culture studies involving caspases (reviewed by
Guo and Hay, 1999;
Los et al., 2001
;
Mendelsohn et al., 2002
) and
Bcl2 family members (Los et al.,
2001
). How Apaf1 and apoptosis affect cell proliferation in the
otic epithelium remains to be clarified. Another open question is why the
phenotypes in brain and ear are so different. It may be because of the fact
that the neuroectodermal cells continue to proliferate by default in the
absence of cell death, whereas the ectodermal cells of placodal origin do not
seem to proliferate by default and might require stimuli and signals from the
neighboring dying cells in order to proliferate normally.
Closure of the otic vesicle does not occur in the absence of both Apaf1 and Bcl2l gene products
Apoptotic cells have been detected in the area of otic vesicle closure in
normal embryos. However, no defects related to the closure were observed in
Apaf1 or Bcl2l single mutant embryos. Interestingly, when
Apaf1 mutation was introduced into the Bcl2l-deficient
background, the otic vesicle closure was incomplete in 100% of the mutants.
These observations suggest that Apaf1 and pro-apoptotic
Bcl2l gene product(s) may have redundant functions in controlling
cell death at the closure site. The function of pro-apoptotic Bcl2l
gene product(s) at the otic vesicle closure site is further supported by the
fact that we observed a reduction, rather than an increase, in cell death at
the otic vesicle closure site in Bcl2l mutant embryos. The
Apaf1-/-/Bcl2l-/- phenotype is the
first example of an otic vesicle closure defect in mouse mutants.
Interestingly, the otic epithelium compartmentalization seems to occur despite
its incomplete closure.
In conclusion, we show that Apaf1-dependent and Bcl-XL-regulated apoptosis is critical for the normal morphological development and the outgrowth of the otic epithelium. Furthermore, we propose that, together with Apaf1, pro-apoptotic Bcl-XL isoform(s) produced from cleavage of Bcl-XL could play a role in otic vesicle closure and in the formation of the posterior semicircular duct.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acampora, D., Merlo, G. R., Paleari, L., Zerega, B.,
Postiglione, M., Mantero, S., Bober, E., Barbieri, O., Simeone, A. and Levi,
G. (1999). Craniofacial, vestibular and bone defects in mice
lacking the Distal-less-related gene Dlx5. Development
126,3795
-3809.
Basañez, G., Zhang, J., Chau, B. N., Maksaev, G. I.,
Frolov, V.A., Brandt, T. A., Burch, J., Hardwick, J. M. and Zimmerberg, J.
(2001). Pro-apoptotic cleavage products of Bcl-xL form cytochrome
c-conducting pores in pure lipid membranes. J. Biol.
Chem. 276,31083
-31091.
Brigande, J. V., Iten, L. E. and Fekete, D. M. (2000). A fate map of chick otic cup closure reveals lineage boundaries in the dorsal otocyst. Dev. Biol. 227,256 -270.[CrossRef][Medline]
Cecconi, F., Alvarez-Bolado, G., Meyer, B. I., Roth, K. and Gruss, P. (1998). Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94,727 -737.[Medline]
Cecconi, F. and Gruss, P. (2001). Apaf1 in developmental apoptosis and cancer: how many ways to die? Cell. Mol. Life Sci. 58,1688 -1697.[Medline]
Clem, R. J., Cheng, E. H., Karp, C. L., Kirsch, D. G, Ueno, K.,
Takahashi, A., Kastan, M. B., Griffin, D. E., Earnshaw, W. C., Veliuona, M. A.
et al. (1998). Modulation of cell death by Bcl-XL through
caspase interaction. Proc. Natl. Acad. Sci. USA
95,554
-559.
Dressler, G. R., Deutsch, U., Chowdhury, K., Nornes, H. O. and Gruss, P. (1990). Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development 109,787 -795.[Abstract]
D'Mello, S. R. (1998). Molecular regulation of neuronal apoptosis. In Current Topics in Developmental Biology, Vol. 39 (ed. R. A. Pedersen and G. P. Schatten), pp. 187-213. London: Academic Press.[Medline]
Fekete, D. M. (1999). Development of the vertebrate ear: insights from knockouts and mutants. Trends Neurosci. 22,263 -269.[CrossRef][Medline]
Fekete, D. M., Homburger, S. A., Waring, T., Riedl, A. E. and
Garcia, L. F. (1997). Involvement of programmed cell death in
morphogenesis of the vertebrate inner ear. Development
124,2451
-2461.
Fujita, N., Nagahashi, A., Nagashima, K., Rokudai, S. and Tsuruo, T. (1998). Acceleration of apoptotic cell death after the cleavage of Bcl-XL protein by caspase-3-like proteases. Oncogene 17,1295 -1304.[CrossRef][Medline]
Guo, M. and Hay, B. A. (1999). Cell proliferation and apoptosis. Curr. Opin. Cell Biol. 11,745 -752.[CrossRef][Medline]
Hakem, R., Hakem, A., Duncan, G. S., Henderson, J. T., Woo, M., Soengas, M. S., Elia, A., de la Pompa, J. L., Kagi, D., Khoo, W. et al. (1998). Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94,339 -352.[Medline]
Hogan, B., Beddington, R., Constantini, F. and Lacy, E. (1994). Manipulating The Mouse Embryo. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Honarpour, N., Du, C., Richardson, J. A., Hammer, R. E., Wang, X. and Herz, J. (2000). Adult Apaf-1-deficient mice exhibit male infertility. Dev. Biol. 218,248 -258.[CrossRef][Medline]
Kluck, R. M., Bossy-Wetzel, E., Green, D. R. and Newmeyer, D.
D. (1997). The release of cytochrome c from mitochondria: a
primary site for Bcl-2 regulation of apoptosis.
Science 275,1132
-1136.
Kuida, K., Haydar, T. F., Kuan, C. Y., Gu, Y., Taya, C., Karasuyama, H., Su, M. S. and Flavell, R. A. (1998). Reduced apoptosis and cytochrome c-mediated caspase activity in mice lacking caspase 9. Cell 94,325 -337.[Medline]
Lang, H., Miller Bever, M. and Fekete, D. M. (2000). Cell proliferation and cell death in the developing chick inner ear: spatial and temporal patterns. J. Comp. Neurol. 417,205 -220.[CrossRef][Medline]
Los, M., Stroh, C., Janicke, R. U., Engels, I. H. and Schulze-Osthoff, K. (2001). Caspases: more than just killers? Trends Immunol. 22,31 -34.[CrossRef][Medline]
Marovitz, W. F., Shugar, J. M. and Khan, K. M. (1976). The role of cellular degeneration in the normal development of (rat) otocyst. Laryngoscope 86,1413 -1425.[Medline]
Marovitz, W. F., Khan, K. M. and Schulte, T. (1977). Ultrastructural development of the early rat otocyst. Ann. Otol. Rhinol. Laryngol. Suppl. 86, 9-28.[Medline]
Marsden, V. S., O'Connor, L., O'Reilly, L. A., Silke, J., Metcalf, D., Ekert, P. G., Huang, D. C. S., Cecconi, F., Kuida, K., Tomaselli, K. J. et al. (2002). Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature 419,634 -637.[CrossRef][Medline]
Martin, P. and Swanson, G. J. (1993). Descriptive and experimental analysis of the epithelial remodellings that control semicircular canal formation in the developing mouse inner ear. Dev. Biol. 159,549 -558.[CrossRef][Medline]
Mendelsohn, A. R., Hamer, J. D., Wang, Z. B. and Brent, R.
(2002). Cyclin D3 activates Caspase 2, connecting cell
proliferation with cell death. Proc. Natl. Acad. Sci.
USA 99,6871
-6876.
Motoyama, N., Wang, F., Roth, K., Sawa, H., Nakayama, K., Nakayama, K., Negishi, I., Senju, S., Zhang, Q., Fujii, S. et al. (1995). Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science 267,1506 -1510.[Medline]
Nicolic, P., Järlebark, L. E., Billet, T. and Thorne, P. R. (2000). Apoptosis in the developing rat cochlea and its related structures. Dev. Brain Res. 119, 75-83.[Medline]
Nishikori, T., Hatta, T., Kawauchi, H. and Otani, H. (1999). Apoptosis during inner ear development in human and mouse embryos: an analysis by computer-assisted three-dimensional reconstruction. Anat. Embryol. 200,19 -26.[CrossRef][Medline]
Noramly, S. and Grainger, R. M. (2002). Determination of the embryonic inner ear. J. Neurobiol. 53,100 -128.[CrossRef][Medline]
Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biol. 2,3005 .1-3005.12.
Represa, J. J., Moro, J. A., Pastor, F., Gato, A. and Barbosa, E. (1990). Patterns of epithelial cell death during early development of the human inner ear. Ann. Otol. Rhinol. Laryngol. 99,482 -488.[Medline]
Salminen, M., Meyer, B. I. and Gruss, P. (1998). Efficient polyA trap approach allows the capture of genes specifically active in differentiated embryonic stem cells and in mouse embryos. Dev. Dyn. 212,326 -333.[CrossRef][Medline]
Salminen, M., Meyer, B. I., Bober, E. and Gruss, P.
(2000). Netrin 1 is required for semicircular canal formation in
the mouse inner ear. Development
127, 13-22.
Sanders, E. J. and Wride, M. A. (1995). Programmed cell death in development. Int. J. Cytol. 163,105 -173.
Scaffidi, P., Misteli, T. and Bianchi, M. E. (2002). Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418,191 -195.[CrossRef][Medline]
Serafini, T., Colamarino, S. A., Leonardo, E. D., Wang, H., Beddington, R., Skarnes, W. C. and Tessier-Lavigne, M. (1996). Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87,1001 -1014.[Medline]
Sher, A. E. (1971). The embryonic and postnatal development of the inner ear of the mouse. Acta Oto-Laryngol. (Suppl.) 285,1 -77.
Takahashi, K., Kamiya, K., Urase, K., Suga, M., Takizawa, T., Mori, H., Yoshikawa, Y., Ichimura, K., Kuida, K. and Momoi, T. (2001). Caspase-3-deficiency induces hyperplasia of supporting cells and degeneration of sensory cells resulting in the hearing loss. Brain Res. 894,359 -367.[CrossRef][Medline]
Veis, D. J., Sorenson, C. M., Shutter, J. R. and Korsmeyer, S. J. (1993). Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75,229 -249.[Medline]
Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai,
J., Peng, T-I., Jones, D. P. and Wang, X. (1997). Prevention
of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science 275,1129
-1132.
Yoshida, H., Kong, Y. Y., Yoshida, R., Elia, A. J., Hakem, A., Hakem, R., Penninger, J. M. and Mak, T. W. (1998). Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94,739 -750.[Medline]
Yoshida, H., Okada, Y., Kinoshita, N., Hara, H., Sasaki, M., Sawa, H., Nagashima, K., Mak, T. W., Ikeda, K. and Motoyama, N. (2002). Differential requirement for Apaf1 and Bcl-XL in the regulation of programmed cell death during development. Cell Death Differ. 9,1273 -1276.[CrossRef][Medline]
Yuan, J. and Yankner, B. A. (2000). Apoptosis in the nervous system. Nature 407,802 -809.[CrossRef][Medline]