RESEARCH ARTICLE 1663
Development 134, 1663-1670 (2007) doi:10.1242/dev.02838
Specific ablation of the transcription factor CREB in
sympathetic neurons surprisingly protects against
developmentally regulated apoptosis
Rosanna Parlato*, Christiane Otto*,†, Yvonne Begus‡, Stephanie Stotz and Günther Schütz§
The cyclic-AMP response element-binding (CREB) protein family of transcription factors plays a crucial role in supporting the survival
of neurons. However, a cell-autonomous role has not been addressed in vivo. To investigate the cell-specific role of CREB, we used as
a model developing sympathetic neurons, whose survival in vitro is dependent on CREB activity. We generated mice lacking CREB in
noradrenergic (NA) and adrenergic neurons and compared them with the phenotype of the germline CREB mutant. Whereas the
germline CREB mutant revealed increased apoptosis of NA neurons and misplacement of sympathetic precursors, the NA neuronspecific mutation unexpectedly led to reduced levels of caspase-3-dependent apoptosis in sympathetic ganglia during the period of
naturally occurring neuronal death. A reduced level of p75 neurotrophin receptor expression in the absence of CREB was shown to
be responsible. Thus, our analysis indicates that the activity of cell-autonomous pro-survival signalling is operative in developing
sympathetic neurons in the absence of CREB.
INTRODUCTION
Cyclic-AMP response element-binding protein (CREB) is a
ubiquitously expressed transcription factor and, together with the
related proteins cAMP-responsive element modulator (CREM) and
ATF-1 (also known as ATF1 – Mouse Genome Informatics), is
activated via cAMP and several other signalling pathways
supporting the survival, growth and plasticity of neurons throughout
development and in adulthood (Lonze and Ginty, 2002).
The first mutation of the Creb gene (also known as Creb1 –
Mouse Genome Informatics) realized in mice, the so-called
CREB␣⌬ mutation, results in a viable hypomorphic allele (Blendy
et al., 1996; Hummler et al., 1994). In contrast to the CREB␣⌬
mutation, a null allele is lethal at birth because of postnatal lung
failure (Rudolph et al., 1998). By conditional mutagenesis the
specific deletion of the Creb gene in neural and glial precursors was
achieved, and massive widespread apoptosis in the embryonic brain
was observed when these mutants were also Crem deficient
(Mantamadiotis et al., 2002). Moreover, specific deletion of the
Creb gene in the postnatal forebrain resulted in selective and
progressive neurodegeneration of the striatum and part of the
hippocampus in the absence of CREM (Mantamadiotis et al.,
2002). Analysis of these mutants also revealed that compensatory
activities exist among these members of the CREB family
(Mantamadiotis et al., 2002).
However, the cell-specific role of CREB in neuronal survival has
not been addressed. We have recently demonstrated by conditional
ablation of CREB in dopaminergic neurons only that the survival of
Department of Molecular Biology of the Cell I, German Cancer Research Center,
D-69120 Heidelberg, Germany.
*These authors contributed equally to this work
†
Present address: Gynecology and Andrology, Schering AG, Berlin, Germany
‡
Present address: Department of Gastroenterology, Hannover Medical School,
Hannover, Germany
§
Author for correspondence (e-mail: [email protected])
Accepted 16 February 2007
dopaminergic neurons is weakly affected and that CREM
upregulation does not contribute to the phenotype (Parlato et al.,
2006).
To investigate the cell-specific role of CREB we used as a model
developing sympathetic neurons. The sympathetic neurons have
been a classical model for the study of the molecular mechanisms
underlying neuronal survival activated by target-derived
neurotrophins (Levi-Montalcini, 1987). During embryonic
development neurons are produced in excess and their survival is
controlled by the availability of target-derived growth factors. In
many areas of the nervous system, programmed cell death is the
predominant mechanism for determining mature neuron number.
The final number of neurons is thus dependent on the balance
between signals leading to either cell death or cell survival. In the
peripheral nervous system this process to select surviving neurons
is, in part, dependent on the nerve growth factor (NGF), acting
through transcriptional regulation of gene expression.
A role for CREB in NGF-dependent survival has been clearly
demonstrated for the sensory ganglia (Lonze et al., 2002). In vitro
experiments performed on postnatal sympathetic neuronal cultures
indicated that NGF-dependent survival requires CREB-mediated
gene expression. These experiments also indicate that BCL-2 (also
known as BCL2 – Mouse Genome Informatics) could be the prosurvival effector of CREB activity (Riccio et al., 1999). However, it
remains to be established whether these in vitro observations reflect
the role played by CREB in vivo in regulating the survival of
sympathetic neurons. Analysis of the neurons in the superior
cervical ganglia (SCG) of CREB knockout mice suggested the
importance of CREB in the survival of sympathetic neurons during
embryonic development (Lonze et al., 2002). However, because the
number of sympathetic neurons is already affected before the
acquisition of NGF-dependence for survival and the CREB protein
is ubiquitously ablated, the crucial role of CREB in mediating
survival of sympathetic neurons (Riccio et al., 1999) could not be
unequivocally established in vivo using CREB knockout mice as a
model (Lonze et al., 2002). Prior to the requirement of NGF, recent
genetic evidence has indicated the importance of other extracellular
DEVELOPMENT
KEY WORDS: CREB, CREM, Sympathetic ganglia, Apoptosis, Mouse
1664 RESEARCH ARTICLE
MATERIALS AND METHODS
Generation and genotyping of mutant mice
A PAC harboring the DBH gene was isolated from the RPCI21 mouse
genomic library. Using ET recombination (Zhang et al., 1998), the coding
sequence of the iCre, followed by the bovine growth hormone
polyadenylation signal, was introduced in-frame with the ATG of the Dbh
gene. The linear insert of 150 kb, carrying the transgenic construct, was
released by NotI digestion and separated by pulse field gel electrophoresis.
The male pronucleus of fertilized C57Bl/6 mice was injected with the DNA
purified by agarase treatment and microdialysis (Schedl et al., 1996).
Transgenic offspring was identified by dot blot and hybridization with a
probe specific for the iCre.
To exclude the possibility that CREM or ATF-1 may compensate CREB,
double-mutant mice were also generated crossing Creb+/fl;DBHCre+/– mice
with Crem+/– or Atf-1+/– mice. The progeny of this first cross was then mated
to yield F2 progeny of interest.
Crebfl/fl Crem–/– DBHCre+/–, abbreviated as CREBDBHCre; CREM–/–, as
well as CrebDBHCre; Atf-1–/– mice are viable. The statistical distribution of
this and other genotypes remains close to the expected values. The analysis
of the genotype was performed as previously described (Mantamadiotis et
al., 2002). iCre PCR primers were the following: forward 5⬘-CTGCCAGGGACATGGCCAGG-3⬘; reverse 5⬘-GCACAGTCGAGGCTGATCAGC-3⬘.
Histology, immunohistochemistry and in situ hybridization
For the detection of ␤-galactosidase activity, embryos were processed as
described elsewhere (Hogan et al., 1994).
For immunohistochemistry (IHC), embryos were fixed in 4%
paraformaldehyde, pH 7.2, overnight, processed for paraffin sections,
sectioned at 7 ␮m and stained with cresyl violet. For cryosections the
samples were treated by 30% sucrose in PBS, embedded in OCT and
sectioned at 20 ␮m. For IHC the following primary antibodies were used:
anti-DBH (rabbit 1:500, DBH12-A; Alpha Diagnostic), anti-Cre (rabbit
1:3000), anti-CREB (rabbit 1:3000), anti-CREM (rabbit 1:500)
(Mantamadiotis et al., 2002), anti-tyrosine hydroxylase (TH) (sheep 1:500,
AB1542; Chemicon), anti-cleaved caspase-3 (Asp175) antibody (rabbit
1:800; Cell Signalling Technology), anti-ATF-1 (rabbit 1:2000) (Bleckmann
et al., 2002), and anti-p75 neurotrophin receptor (p75NTR) (rabbit 1:2000,
AB1554; Chemicon). The sections were incubated in citrate buffer, pH 6.0,
and boiled in a microwave oven. The primary antibodies were incubated
overnight at 4°C. Biotin-conjugated secondary antibody was diluted 1:400
in PBS and detection was performed using the avidin-biotin system (Vector
Laboratories) with the VECTOR peroxidase kit. The staining was developed
with DAB and H2O2 (Sigma) or with HistoGreen (Linaris). For double
immunolabeling with anti-TH and anti-activated caspase-3 antibodies, the
activity of the first antibody was blocked by the Avidin/Biotin blocking kit
(Vector Laboratories). Sections were stained as described for a single
antigen, and the second staining was performed with DAB, giving a blue
precipitate (Sigma).
Whole-mount TH immunostaining was performed as previously
described (Enomoto et al., 2001).
Non-radioactive in situ hybridization was performed on paraffin sections
as previously described (Parlato et al., 2004). The expression of p75NTR
mRNA was analyzed by using two riboprobes recognizing the p75NTR
intracellular domain or the extracellular domain, respectively, as designed by
McQuillen et al. (McQuillen et al., 2002). The expression pattern obtained
with both riboprobes was similar, and therefore we have shown only the
experiments performed with the p75NTR intracellular domain riboprobe.
Cell counts and statistical analysis
After caspase-3 immunolabeling, the sections were counterstained with
Nuclear Fast Red (Vector). Cell counts were performed at 40⫻
magnification in bright field. Clearly identified caspase-3-positive cells
characterized by brown colour were counted as positive in sections
containing the SCG and the stellate ganglia for both control and mutant
mice. For area and volume measurements, the IMAGE J program was used.
The average number of caspase-3-positive cells per mm2 was calculated for
every fourth section per ganglion, spanning the entire SCGs, in at least four
sections per side, because both SCGs were analyzed. Values shown are
mean±s.e.m. for at least four to five mice for each genotype. The volume of
the SCG reported is the mean±s.e.m. for both SCGs in at least four to five
mice per genotype. The total number of neurons per ganglia was determined
by counting the neurons with visible nuclei in every fourth section. The total
counts were quadrupled to calculate the total number of neurons. Values
shown are mean±s.e.m. Statistical significance was analyzed using a
homoscedastic Student’s t-test. Values were considered significantly
different with *P<0.05 and ***P<0.001.
The average number of caspase-3-positive cells per section was also
calculated for each animal. In this case, values are mean±s.e.m. for 12-15
sections per animal (both SCGs were analyzed in at least four to five mice
of each genotype) (data not shown).
RESULTS
Generation of CREB-deficient mice in NA neurons
In order to study whether CREB-mediated signalling controls
survival of sympathetic neurons, we used the Cre/loxP system to
obtain mice with selective loss of CREB in these neurons. To
faithfully drive the expression of the Cre recombinase, we used the
regulatory regions of the DBH gene contained in a PAC clone
(DBHCre) (Fig. 1A). To analyze Cre recombinase selectivity,
DBHCre mice were crossed to the Rosa26 reporter line, in which
recombination results in expression of the ␤-galactosidase gene
(Soriano, 1999). ␤-galactosidase activity was revealed at E11.5 (Fig.
1B) in different regions of the developing sympathetic chain (Fig.
1B), as depicted in the areas (i), (ii) and (iii). Positive staining is also
present in a region dorsolateral to the fourth ventricle where the
precursors of the locus coeruleus (LC), the major NA neurons of the
brain, are located (data not shown).
Mice in which exon 10 of the Creb gene is flanked by loxP sites
(floxed Creb allele; Crebfl) (Mantamadiotis et al., 2002) were
crossed to DBHCre mice to generate Crebfl/fl;DBHCre mutants
(CrebDBHCre). CREB immunoreactivity is already strongly reduced
by E11.5 in the sympathetic chain of CrebDBHCre mutants (data not
shown). Some differences were found in the sympathetic chain at
E11.5, because neurons located in the rostral part of the embryo
show lower CREB expression compared with the caudal part. This
DEVELOPMENT
cues, such as the glial cell line-derived neurotrophic factor (GDNF)
family member artemin (Honma et al., 2002), in sympathetic axon
outgrowth and directed neuronal migration via GFR␣3 (Nishino et
al., 1999) and RET (Enomoto et al., 2001). Thus, it is of great
interest to analyze whether CREB-dependent signalling is of crucial
importance not only for NGF-dependent survival, but also at earlier
stages in the development and migration of sympathetic ganglia.
In order to investigate the role of CREB in developing
sympathetic ganglia, we generated mouse mutants in which the Creb
gene is deleted specifically in noradrenergic (NA) and adrenergic
neurons of the central and peripheral nervous system (Crebfl/fl;
DBHCre mice, abbreviated as CrebDBHCre) in a Crem- or Atf-1-null
genetic background. This specific mutation was made possible
because we had developed transgenic mice faithfully expressing the
Cre recombinase in cells under the control of the gene for dopamine␤-hydroxylase (DBHCre) using the PAC technology, which allows
position-independent expression of the transgene (Casanova et al.,
2001; Parlato et al., 2006; Wintermantel et al., 2002). This Cre line
has also been used for the specific inactivation of the gp130
signalling in sympathetic neurons (Stanke et al., 2006).
The analysis of the specific ablation of CREB in comparison with
the CREB knockout mice provides unexpected new insights into the
cell-autonomous role of CREB, and demonstrates that loss of CREB
unexpectedly results in neuroprotection.
Development 134 (9)
Fig. 1. Generation of a CREB mutation selective for sympathetic
neurons. (A) Schematic representation of the DBHiCre PAC used for
transgenesis. The linear insert of 150 kb carrying the transgene
construct contains approximately 100 kb upstream of the ATG of the
Dbh gene. (B) Detection of DBHCre activity at E11.5 in the reporter
mouse line ROSA26 by whole-mount ␤-galactosidase staining in
sagittal sections at different levels of the sympathetic chain, from rostral
to caudal (i), (ii) and (iii). Adjacent sagittal sections from E12.5 control
(Crebfl/fl) and mutant (CrebDBHCre) embryos are stained with anti-DBH
(C,F), anti-Cre (D,G) and anti-CREB (E,H) antibodies. At E12.5, DBH
expression characterizes sympathetic precursors in control (C) and
mutant (F) embryos. In control Cre-negative precursors, CREB protein is
present (D,E), whereas Cre-positive cells lose CREB immunoreactivity in
the conditional mutants (G,H). Scale bar: 2 mm in B; 130 ␮m in C-H.
observation is consistent with the different timing in the maturation
of the sympathetic chain, which follows rostro-caudal patterning
(Hagedorn et al., 2000).
A more dramatic decrease in CREB immunoreactivity is evident
in CrebDBHCre mice at E12.5 (Fig. 1E,H). The pattern of CREB loss
is consistent with the expression of Cre (Fig. 1D,G), which
reproduces the expression of DBH (Fig. 1C,F). At E17.5, CREB
immunoreactivity is lost in most cells of the SCG, presumably
neurons, but it is preserved in other cell types, as shown in Fig. 2A,B.
The analysis of the SCG at E17.5 reveals no major alterations in size
and morphology of NA neurons in the CrebDBHCre mutants (Fig.
2C,D). Unlike the CREB germline mutants (Creb–/–), the CrebDBHCre
conditional mutants survive after birth without showing a reduced
lifespan. We analyzed the sympathetic projections of the sympathetic
postganglionic axons from the SCG by whole-mount IHC with TH
antibody at P2. As shown in Fig. 2E,F, the cutaneous sympathetic
innervation of the eye is not impaired in the CrebDBHCre mutants.
Germline deletion of Creb results in aberrant
morphology of the sympathetic chain
Because it is possible that the other member of the CREB family
expressed in the central nervous system, CREM, compensates for
CREB loss, we decided to generate mice that are CrebDBHCre;
RESEARCH ARTICLE 1665
Fig. 2. CrebDBHCre mutants show no gross morphological
abnormalities in the sympathetic ganglia. Sagittal sections from
control (Crebfl/fl) and mutant (CrebDBHCre) embryos at E17.5 are stained
with anti-CREB antibody (A,B). CREB immunoreactivity is indicated in
the SCG (enclosed area) by arrows in neurons, and by arrowheads in
other morphologically distinct cells. Nissl staining reveals no major
differences between control (C) and mutant (D). Whole-mount
immunohistochemistry with TH antibody of P2 control (E) and
CrebDBHCre (F) animals reveals no major deficits in ophthalmic
projections (arrows). e, eye. Scale bar: 100 ␮m in A,B; 50 ␮m in C,D;
500 ␮m in E,F.
CREM–/–. These mutants also survive after birth and show no
reduced lifespan or behavioural anomalies. The analysis of
sympathetic ganglia, performed by IHC with an antibody against
TH, at E15.5, a developmental stage independent of NGF for
survival of sympathetic neurons, reveals that the SCG and the
stellate ganglion in CrebDBHCre; Crem–/– (Fig. 3B,D) are properly
shaped and placed in comparison to control littermates (Fig.
3A,C). At the same stage, in Creb-null mice, the overall
organization of the sympathetic ganglia is severely affected, as
shown in Fig. 3F. At E17.5, a smaller SCG in the Creb-null
mutants, located in the cervical region (Fig. 3G,H, area circled in
red), and a bigger stellate ganglion in the thoracic region (Fig.
3G,H) is seen.
Specific ablation of Creb protects against
developmentally regulated apoptosis
To analyze the physiological relevance of the absence of
CREB specifically in sympathetic neurons, we decided to
analyze apoptotic neurons in the SCG and the stellate ganglia at
different developmental stages in controls, Creb-null and
CrebDBHCre; Crem–/– mutants (Fig. 4). Although we concentrate
our studies on the SCG, which, because of its large size, its
accessibility and its vascular supply has been classically used as
DEVELOPMENT
CREB role in developing sympathetic ganglia
model system to study survival of sympathetic neurons, similar
observations were also made in the stellate ganglia (data not
shown).
Naturally occurring cell death in sympathetic ganglia starts in
mice at E16-E17 (Coughlin and Collins, 1985), and indeed in mice
that lack NGF, neuronal loss can be detected by E17.5 and at P0.
This is associated with a 90% decrease in SCG volume, indicating
that NGF action on sympathetic neurons takes place in this time
window (Crowley et al., 1994; Francis and Landis, 1999). At
E17.5, as revealed using IHC for activated caspase-3 in
combination with the specific marker TH, the analysis of control
embryos reveals the presence of apoptotic cells in the SCG. In
CREB-null mice apoptotic cells are even more strongly
represented (Fig. 4B,E). In CREB-null mice, it is not possible to
clearly distinguish the SCG from the stellate ganglion, therefore
we have measured the total volume of the SCG and stellate
ganglia, identified by TH staining. Although no changes are
revealed in the total volume of stellate ganglia and SCG between
Fig. 3. Defects in migration of SCG cells in Creb-null mice.
Parasagittal sections of E15.5 mouse embryos immunostained with TH
antibody show the SCG normally shaped and located in proximity to
the inner ear (asterisk) in control (A) and CrebDBHCre; Crem–/– mutants
(B), but not in Creb–/– mutants (F). The stellate ganglion is located in the
thoracic region in the CrebDBHCre; Crem–/– mutant (D) and in the
respective control littermate (C) as well as in the control littermate of
Creb–/– (E). However, in Creb–/– it extends more rostrally (F). At E17.5 a
similar pattern is found in Creb–/– showing less sympathetic neurons in
the area of the SCG (H, circled area) and more in the area of the
stellate ganglion in comparison with control (G). Black arrowhead
indicates the tubercle of the first rib, used as a positional reference to
compare the position of the stellate ganglia. Scale bar: 300 ␮m in A-F;
600 ␮m in G,H.
Development 134 (9)
controls and mutants at E17.5 (data not shown), it is well possible
that the increased apoptosis observed in the Creb-null mice at this
stage would postnatally result in a smaller sympathetic ganglia.
Surprisingly, in CrebDBHCre; Crem–/– mutants, in contrast to CREBnull mice, there is no increased apoptosis (Fig. 4C,F), rather a
decrease in the number of caspase-3-positive cells in the SCG, as
Fig. 4. Survival of sympathetic neurons in absence of CREB.
Immunohistochemistry with an antibody recognizing activated caspase3 (brown) is used to analyze survival of sympathetic neurons in the SCG
at E17.5, and anti-TH antibody (blue) is used to identify the region of
interest in controls (A,D), Creb–/– (B,E) and CrebDBHCre; Crem–/– mutants
(C,F). Representative sections are shown in A-F. Red arrows indicate
examples of activated caspase-3-positive cells. (G) Quantitative analysis
of apoptotic cells reveals reduced levels of apoptosis in sympathetic
neurons of CrebDBHCre; Crem–/– mutants (abbreviated as M) at E17.5 in
comparison with controls (abbreviated as C). At postnatal stages P0/P2
the level of apoptosis is reduced in control pups and does not change in
CrebDBHCre; Crem–/– mutants. (H) The size of the SCG in CrebDBHCre;
Crem–/– mutants is similar between controls and mutants at E17.5. At
P0/P2 the size of the SCG in CrebDBHCre; Crem–/– mutants is significantly
larger than in controls. The mean±s.e.m. for both SCG in at least four
to five mice of each genotype are shown. Values are considered
significantly different with *P<0.05 and ***P<0.001 compared with
controls. Scale bar: 250 ␮m in A-C; 40 ␮m in D-F. Asterisk indicates the
inner ear.
DEVELOPMENT
1666 RESEARCH ARTICLE
CREB role in developing sympathetic ganglia
summarized in Fig. 4G. In order to exclude the possibility of an
earlier onset of apoptosis in CrebDBHCre; Crem–/–, we searched for
the presence of apoptotic cells at E15.5, but undetectable levels of
apoptosis in the SCG characterize this early stage without
significant differences between genotypes (data not shown).
Because the CrebDBHCre; Crem–/– mutants, in contrast to the
germline CREB mutation, are not postnatally lethal, we
determined the number of caspase-3-positive cells at postnatal
stages P0/P2 (n=4-5 per genotype). We observe a reduced level of
apoptosis in the control SCG in comparison with E17.5 (Fig. 4G),
with no changes in the CrebDBHCre; Crem–/– mutants. When we
measured the total volume of the SCG at E17.5 there were no
significant differences between controls and CrebDBHCre; Crem–/–
mutants (Fig. 4H). At P0/P2 we found that there is an overall
increase in the size of the SCG, consistent with the reduced
number of apoptotic cells occurring at E17.5 in CrebDBHCre;
Crem–/– (Fig. 4H). The number of SCG neurons at P2 is
significantly increased (control: 11014±1611; CrebDBHCre; Crem–/–
mutants: 19661±1905, P<0.05). These results clearly indicate that
the specific loss of CREB in developing sympathetic neurons not
only does not lead to decreased neuronal survival, but
unexpectedly has a protective effect against developmentally
regulated apoptosis.
RESEARCH ARTICLE 1667
(Bleckmann et al., 2002; Mantamadiotis et al., 2002), neither
CREM nor ATF-1 play a crucial role in the survival of sympathetic
neurons.
p75NTR expression in sympathetic neurons
depends on CREB
To shed light on the molecular mechanisms underlying the reduced
levels of apoptosis observed in the conditional CREB mutants, we
reasoned that the expression of crucial factors promoting
developmental death of sympathetic neurons might be CREBdependent. It is well established that the p75NTR plays an important
role in promoting apoptosis of sympathetic neurons lacking
appropriate levels of target-derived neurotrophins, such as NGF
(Bamji et al., 1998; Majdan and Miller, 1999). Interestingly, the
phenotype of the CREB conditional mutants is reminiscent of the
phenotype of p75NTR–/– mice, also showing an increase in the relative
number of sympathetic neurons (Brennan et al., 1999).
Therefore, we have analyzed by in situ hybridization the
expression of several potential CREB target genes involved in the
p75NTR-mediated signalling in sympathetic ganglia of control and
Fig. 5. Neither CREM nor ATF-1 influence survival of sympathetic
neurons. Quantitative analysis of apoptotic cells reveals reduced levels
of apoptosis in sympathetic neurons of CrebDBHCre, CrebDBHCre; Crem+/–,
CrebDBHCre; Crem–/– and CrebDBHCre; Atf-1–/– mutants at E17.5 in
comparison with the respective controls. The mean±s.e.m. for both
SCGs in at least three mice of each genotype are shown. Values are
considered significantly different with *P<0.05 compared with controls.
Fig. 6. p75NTR expression in sympathetic neurons is dependent on
CREB. Non-radioactive in situ hybridization with a riboprobe specific
for the p75NTR on representative sagittal sections from control (A,C)
and CrebDBHCre; Crem–/– (B,D) showing the SCG (A,B) and basal
cholinergic neurons (C,D) at E17.5. Asterisk indicates the inner ear.
Immunohistochemistry with an antibody recognizing the p75NTR protein
is used to analyze protein expression in the SCG at E17.5 in controls (E)
and in CrebDBHCre; Crem–/– (F). Scale bar: 250 ␮m in A-D; 40 ␮m in E,F;
20 ␮m in insets.
DEVELOPMENT
Loss of CREB is sufficient to inhibit
developmentally regulated apoptosis
In order to establish the role of CREM or ATF-1 in survival of
sympathetic neurons, we have compared the number of caspase-3positive cells at E17.5 (n=3 per genotype) in CrebDBHCre mutants
as well as in CrebDBHCre; Crem+/–, CrebDBHCre; Crem–/–, and
CrebDBHCre; Atf-1–/– (Fig. 5). Independent of the presence of the
CREM or ATF-1 alleles, we observed a similar decrease in the
number of apoptotic cells in all the mutants in comparison with the
respective controls. These data indicate that loss of CREB alone
accounts for the decreased apoptosis observed in the conditional
mutant embryos, as already suggested by lack of CREM and
ATF-1 immunoreactivity at this developmental stage in
sympathetic ganglia (data not shown). Unlike in other cell types
1668 RESEARCH ARTICLE
Development 134 (9)
CrebDBHCre; Crem–/– mutants at E17.5 (Fig. 6). At this
developmental stage, we found that in the conditional mutants (Fig.
6B) the levels of p75NTR mRNA are much lower than in control
embryos (Fig. 6A), whereas they are unaltered in other regions not
affected by the mutation, such as the cholinergic neurons of the basal
forebrain (Fig. 6C,D and data not shown). The reduced level of
p75NTR protein in the CrebDBHCre; Crem–/– mutants (Fig. 6F) in
comparison with control littermates (Fig. 6E) probably accounts for
the inhibition of developmentally regulated apoptosis observed in
sympathetic ganglia of the conditional CREB mutant.
Germline loss of CREB results in misplacement
and reduced survival of sympathetic neurons
The results presented in our study are summarized in Fig. 7 by a
schematic model of the sympathetic neuron development in wild
type and in the different CREB mutants analyzed here. Developing
wild-type neurons successfully reaching their targets survive in the
presence of an optimal supply of pro-survival factors. Here, we
oversimplify considering that NGF, secreted by the targets of
sympathetic ganglia during the period of target competition, plays
a major role in defining the final number of surviving sympathetic
neurons. In Creb–/– mutants, CREB expression is lost in
developing neurons, as well as in target cells. Loss of CREB in
target cells may be primarily responsible for the migrational deficit
of the SCG neurons, and hence responsible for the increased
apoptosis in sympathetic neurons in the absence of proper survival
signals originating in target cells. Developing sympathetic
neurons, prior to NGF action, require migrational cues to establish
their final position (Glebova and Ginty, 2005). Interestingly, gene
targeting studies revealed that mice deficient in RET (Enomoto et
al., 2001) or its cofactor GFR␣3 (Nishino et al., 1999) or the ligand
artemin exhibit severe defects in the SCG (Honma et al., 2002)
during the period from E12.5 to E13.5. As in Creb-null mice, the
SCG is found caudal to its normal location in all these mutants,
because neuronal precursors of the sympathetic system fail to
migrate and to project axons properly. These primary deficits lead
to mis-routing of sympathetic nerve trunks and accelerated cell
death of sympathetic neurons later in development. Because
CREB phosphorylation is not only driven by NGF, but also by
GDNF via its receptor tyrosine kinase RET involving the Ras/ERK
pathway for activation (Hayashi et al., 2000), this signalling
Fig. 7. Schematic representation of the sympathetic neurons in
wild type and in the different CREB mutants. Survival of
developing neurons depends on availability of NGF produced by target
organs (black dots). Neurons not receiving enough neurotrophin
undergo apoptotic cell death (broken line). Black and white nuclei
indicate the presence or absence of CREB. In Creb–/– mutants CREB
expression is lost in the developing neurons and also in the target cells,
where CREB may be required to express molecular cues prior to
neurotrophin dependence. Loss of CREB in sympathetic neurons and
target cells results in partial misplacement of the neurons and in
increased neuronal death, possibly exacerbated by the reduced
neurotrophic supply. In the conditional mutants, CREB expression is lost
only in noradrenergic neurons of sympathetic ganglia, resulting in an
increased number of surviving neurons. The neurotrophic supply from
target organs is probably unaffected. This scenario indicates cell
autonomous pro-survival mechanisms operative in the absence of
CREB.
pathway could be affected by loss of CREB. The fact that in the
conditional mutant, the SCG is correctly positioned, despite early
loss of CREB, strongly suggests that CREB expression is required
in cells other than neurons for proper development of sympathetic
ganglia. Because in null mutants CREB ablation also occurs in
cells other than neurons, extraneuronal cues may depend on CREB
for their expression.
A cell-autonomous role of CREB in survival of
sympathetic neurons is indicated by the
conditional mutant
In the sympathetic ganglia of the conditional mutants lacking CREB
only in NA neurons, we evaluate the cell-autonomous role of CREB
activity in pro-survival and pro-apoptotic pathways, because the
neurotrophic supply from target organs is probably unaffected.
Previous work performed on postnatal sympathetic neuronal
cultures in which CREB-mediated gene expression is abolished by
the use of a dominant-negative protein indicates that all three CREB
family members contribute to the survival of sympathetic neurons
(Riccio et al., 1999).
In the present study we focused on developmentally regulated cell
death and we observed that in the conditional CREB mutants lacking
either CREM or ATF-1, this process is inhibited, as evidenced by the
reduced number of activated caspase-3-positive cells in sympathetic
ganglia. Despite what has been previously shown in other CREB
mutants regarding the possibility of compensation by other family
members (Bleckmann et al., 2002; Mantamadiotis et al., 2002), loss
of CREB and CREM or ATF-1 does not result in a more severe
impairment of cell survival (Fig. 5). Although neither CREM nor
ATF-1 is expressed in sympathetic ganglia from controls and
conditional mutants at E17.5 (data not shown), we cannot rule out
the possibility that inactivation of all three transcription factors
DEVELOPMENT
DISCUSSION
The specific role of CREB-dependent gene expression in survival
of specific populations of neurons is so far poorly understood. Using
the Creb germline mutation along with the conditional line lacking
CREB only in NA and adrenergic neurons, we addressed the
question of whether CREB-mediated transcriptional activity is
necessary for the survival of restricted neuronal subtypes, using
developing sympathetic ganglia as a model system.
This analysis revealed that increased cell death is associated with
misplacement of sympathetic ganglia in the CREB germline mutant.
Because both effects are not observed in the conditional mutant,
CREB expression in cells other than sympathetic neurons is required
for neuronal survival and migration. The characterization of the
conditional mutant reveals a novel distinct effect in neuronal
survival upon loss of CREB. Indeed, reduced levels of
developmentally regulated apoptosis are found in the sympathetic
ganglia, resulting in an increased number of sympathetic neurons.
This effect is correlated with reduced activation of caspase-3 and
downregulation of the p75NTR, a signal mediator necessary for
apoptosis in sympathetic neurons.
would result in increased cell death. The generation of transgenic
mice expressing a dominant-negative CREB protein exclusively in
sympathetic neurons could represent a valuable tool to address this
issue.
To our knowledge, this is the first example of mutants in which
CREB ablation leads to protection against cell death. Several
hypotheses can be taken into account to explain this observation. An
imbalance between pro-apoptotic signals and pro-survival signals
during such a crucial time-window could result in an increased
number of surviving sympathetic neurons. It has been indicated that
CREB may regulate a pro-survival factor, such as Bcl-2 (Lonze and
Ginty, 2002; Riccio et al., 1999). However, although in vitro
experiments established that Bcl-2 is an important regulator of
survival of sympathetic neurons after NGF deprivation (Greenlund
et al., 1995), inactivation of Bcl-2 itself in mouse mutants
(Michaelidis et al., 1996) did not result in increased death of
sympathetic neurons during naturally occurring cell death starting
at E16-E17 in mice (Coughlin and Collins, 1985).
Interestingly, the phenotype of the CREB conditional mutants is
reminiscent of the phenotype of Bdnf–/– mice (Bamji et al., 1998),
showing an increase in the relative number of sympathetic neurons.
The brain-derived neurotrophic factor (BDNF) is a wellcharacterized CREB target (Shieh and Ghosh, 1999; Shieh et al.,
1998; Tao et al., 1998). This neurotrophin may play a dual role in the
fate of developing neurons, either promoting their survival or their
apoptotic death. In sympathetic neurons, BDNF signalling may
inhibit axonal growth and neuronal survival through the p75NTR,
because its receptor TrkB is not expressed in sympathetic neurons
(Bibel and Barde, 2000). In vitro experiments indicate that, when
sympathetic neurons are exposed to suboptimal survival signals,
activation of p75NTR by BDNF leads to neuronal apoptosis. Although
p75 signalling mechanisms remain poorly understood, loss of such a
mechanism during the period of cell death could explain the
increased number of sympathetic neurons observed in the Bdnf–/–
mice (Bamji et al., 1998) and in p75NTR–/– mice (Brennan et al., 1999).
The similarity between phenotypic alterations suggests a correlation
between CREB and BDNF/p75NTR signalling. Interestingly, the
p75NTR gene is included, among other signalling molecules, in a
comprehensive study aiming to identify CREB targets by an
approach based on chromatin immunoprecipitation and a
modification of SAGE (Impey et al., 2004). Although the functional
role of CREB in regulating p75NTR gene expression has not been
demonstrated, our finding that lower levels of p75NTR expression are
shown in developing sympathetic neurons of the conditional CREB
mutants, indicates that CREB is required for p75NTR expression.
Consequently, lower levels of p75NTR expression leads to decreased
apoptosis, resulting in enlarged sympathetic ganglia after birth.
In summary, we conclude that CREB expression in cells other
than sympathetic neurons is required for proper shaping of the
sympathetic chain and for controlling neuronal survival, as
illustrated by the comparison between germline and conditional
Creb mutants. Indeed, loss of CREB in developing sympathetic
neurons neither affects their position nor impairs their survival.
Unexpectedly, loss of CREB exclusively in developing sympathetic
neurons results in a protective effect against developmentally
regulated apoptosis because of downregulation of p75NTR
expression.
We thank H. Rohrer and D. Ginty for critical reading of the manuscript and
K. Unsicker for helpful discussions and advice. This work was supported by the
Deutsche Forschungsgemeinschaft through Collaborative Research Centres
SFB488 and SFB636, FOR Ot 165/2-2, GRK 791/1.02, and Sachbeihilfe Schu
51/7-2, by the Fonds der Chemischen Industrie, the European Union through
RESEARCH ARTICLE 1669
grants QLG1-CT-2001-01574 and LSHM-CT-2005-018652 (CRESCENDO), the
Bundesministerium für Bildung und Forschung (BMBF) through NGFN grants
FZK 01GS01117, 01GS0477 and KGCV1/01GS0416, German-Polish
cooperation project 01GZ0310 and project number 0313074C (Systems
biology).
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1670 RESEARCH ARTICLE