First published online 11 December 2002
doi: 10.1242/jcs.00223
Programmed cell death in the developing inner ear is balanced by nerve growth factor and insulin-like growth factor I
Laura M. Frago1,*,
Susana Cañón1,
Enrique J. de la Rosa2,
Yolanda León1,3,
,
and
Isabel Varela-Nieto1,
1 Instituto de Investigaciones Biomédicas "Alberto Sols",
Consejo Superior de Investigaciones Científicas-Universidad
Autónoma de Madrid (CSIC-UAM), Arturo Duperier 4, 28029 Madrid,
Spain
2 Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006
Madrid, Spain
3 Departamento de Biología (Fisiología Animal), Universidad
Autónoma de Madrid, Carretera de Colmenar km. 15, Cantoblanco, 28049
Madrid, Spain
* Present address: Unidad de Investigación, Departamento de
Pediatría-UAM, Hospital Universitario Niño Jesús, Av.
Menéndez Pelayo 65, 28009 Madrid, Spain
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Fig. 1. IGF-I protects otic vesicles from NGF-induced cell death. Apoptotic cell
death was visualised by TUNEL in cultured otic vesicles. Otic vesicles were
isolated from HH18 chicken embryos, made quiescent and cultured for 8 hours in
serum-free medium (A, control), 4 nM NGF (B), 5 µM C2-Cer (C) or
1 nM IGF-I (D) or the combination of IGF-I with NGF (E) or C2-Cer
(F). Compiled projections of the whole otic vesicle are shown. All otic
vesicles have the same orientation: D (dorsal), A (anterior), cvg
(cochleovestibular ganglion). The images shown are representative of at least
three different experiments, using five otic vesicles by condition. Bar, 100
µm.
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Fig. 2. p75NTR is expressed in the developing inner ear. (A)
p75NTR mRNA expression was detected by whole mount in situ
hybridization in HH18 chicken embryos. Arrowheads point to major patches of
p75 expression, within the otic epithelium and in the CVG (cvg). The
microphotograph shows a 60 µm parasagittal vibratome section. At least 12
embryos were sampled in three different experiments. (B) Immunolocalization of
p75NTR protein was positive in the otic epithelium and in the CVG
(arrowheads). The microphotograph shows a 25 µm parasagittal section from
an HH18 chicken embryo. At least 10 embryos were sampled in three different
experiments. (C,D) Detection of apoptotic cells by TUNEL labelling in a 25
µm parasagittal cryostat section from an HH18 embryo. D shows a
magnification of the CVG area. (E) 2.5 µm optic section of an otic vesicle
cultured in the presence of 4 nM NGF. Apoptotic positive cells were visualised
by TUNEL labelling. C and E arrowheads point to apoptotic cells in the otic
epithelium and CVG. C and E microphotographs are representative of at least
three different experiments performed in triplicate. A, anterior; D, dorsal;
cvg marks the CVG area. Bar, 100 µm (A, B, C and E), 50 µm (D).
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Fig. 3. p75NTR and caspases are involved in NGF-induced cell death in
the otic vesicle. Apoptotic cell death was visualised by TUNEL. Otic vesicles
were isolated from HH18 chicken embryos, made quiescent and cultured for a
further period of 8 hours in serum-free medium (A, Control) or 4 nM NGF (B)
alone or in combination with 2 µg/ml anti-NGF antibody (C), (1:100) 9651
anti p75NTR antibody (D) or caspase inhibitors, 25 µM zVAD (F)
and 75 µM DEVD (H), as indicated in the panels. Explants were pre-treated
for 30 minutes with the antibodies or caspase inhibitors. All the otic
vesicles have the same orientation: D (dorsal); A (anterior); cvg
(cochleovestibular ganglion). Compiled projections of the whole otic vesicle
are shown. Representative microphotographs of three independent experiments
using five otic vesicles by condition are shown. Bar, 100 µm.
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Fig. 4. Modulation of NGF-induced ceramide accumulation and PARP hydrolysis. (A)
Modulation of ceramide accumulation by NGF. Five HH18 otic vesicles/per
datapoint were labelled with 25 µCi/ml of [3H]palmitic acid for
24 hours as described in Materials and Methods. Otic vesicles were then
carefully washed and incubated for 3 hours without additives (control) or with
4 nM NGF alone or in combination with 1 nM IGF-I, 2 µg/ml anti-NGF
antibody, 9651 anti-p75NTR antibody (1:100), 25 µM zVAD or 75
µM DEVD. Ceramide accumulation was measured as described in Materials and
Methods. Results are expressed as mean±s.e.m. of at least five
independent experiments performed in triplicate. Statistical significance
estimated by ANOVA was as follows *P<0.005 versus
control, #P<0.005 versus NGF. (B) NGF-induced cell death triggers
PARP-hydrolysis. Hydrolysis of native poly-ADP-ribose polymerase (p116-PARP)
produces a fragment of 85 kDa (p85-PARP). Quiescent HH18 otic vesicles (8 otic
vesicles per data point) were incubated for 16 hours either in serum-free
medium (Control) or in the presence of 4 nM NGF alone or in combination with 1
nM IGF-I, 2 µg/ml anti-NGF antibody, 9651 (1:100) anti-p75NTR
antibody, 25 µM zVAD or 75 µM DEVD. Incubation with the non-blocking
p75NTR antibody or agonist vehicles had no effect on PARP induction
(data not shown). Otic vesicle lysates were analysed by western blotting with
an anti-PARP antibody that recognizes both the intact and the proteolysis
fragment. A representative blot of one of three independent experiments is
shown. Densitometric quantification is shown as bars. Statistical significance
estimated by ANOVA was as follows *P<0.005 versus
control, #P<0.005 versus NGF. (C) C2-Cer-induced cell
death also triggers PARP-hydrolysis. Otic vesicles were incubated in
serum-free medium (Control) or 5 µM C2-Cer alone or in
combination with 1 nM IGF-I, 25 µM zVAD or 75 µM DEVD. Otic vesicle
lysates were analysed by western blotting with an anti-PARP antibody as above.
A representative blot of one of three independent experiments is shown.
Densitometric quantification is shown as bars. Statistical significance
estimated by ANOVA was as follows *P<0.005 versus
control, #P<0.005 versus C2-Cer.
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Fig. 5. IGF-I increases Akt phosphorylation and PCNA levels. (A) Modulation of
NGF-induced ceramide accumulation by R59949. Ceramide accumulation was
measured as described in Materials and Methods. Otic vesicles were incubated
for 3 hours without additives (Control), 1 nM IGF-I, 1 µM R59949 or with 4
nM NGF alone or in combination with IGF-I and R59949. Statistical significance
estimated by ANOVA was as follows *P<0.005 versus
control, #P<0.005 versus NGF. (B) Quiescent HH18 otic vesicles
were incubated for 30 minutes with 1 nM IGF-I, 4 nM NGF, 5 µM
C2-Cer, 25 µM Cer-1-P or combinations of these factors for the
determination of Akt or for 24 hours to measure PCNA levels. When indicated, a
30 minute pre-treatment with 1 µM R59949 was performed. Otic vesicle
lysates were analysed by western blotting for total Akt levels, for the active
phosphorylated form of Akt (Ser473 P-Akt) and for PCNA levels. Representative
blots are shown. The lower panel shows densitometric quantification values of
activated Akt (Akt-P) referred to total Akt levels (grey bars) and of PCNA
(dark bars). Control cultures were taken as 100. Values are given as
mean±s.e.m. of independent experiments performed in duplicate. The
statistical significance between incubations under different conditions
estimated by ANOVA was as follows: *P<0.005 versus
control, #P<0.001 versus IGF-I. R59949 plus IGF-I and NGF or
C2-Cer were statistically significant (P<0.001) with
respect to their equivalent R59949-untreated conditions.
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Fig. 6. NGF signalling through p75 in the otic vesicle. A proposed scheme showing
the intracellular signalling pathways activated by NGF and IGF-I and how they
balance cell death. Arrows denote facilitating action whereas crossbars
indicate inhibitory influence.
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© The Company of Biologists Ltd 2003
