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First published online 24 July 2008
doi: 10.1242/dev.023960
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1 Department of Pathology, Northwestern University, Chicago, IL 60611,
USA.
2 Department of Neurology, Northwestern University, Chicago, IL 60611,
USA.
3 Division of Neuropathology, Northwestern University, Chicago, IL 60611,
USA.
* Author for correspondence (e-mail: warren{at}northwestern.edu)
Accepted 23 June 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Egr3, Sympathetic, Ptosis, Neurotrophin, Physiology, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and Ret
(Enomoto et al., 2001)
tyrosine kinase receptors, as well as through guidance molecules such as
semaphorin 3A (Kawasaki et al.,
2002). In addition, specification of the noradrenergic phenotype
of post-migratory sympathoadrenal precursors depends upon diffusible factors
such as bone morphogenetic proteins (BMPs) and transcriptional regulators such
as Mash1, Phox2a, Phox2b, Hand2 (dHand) and Gata3 (for a review, see
Goridis and Rohrer, 2002;
Howard, 2005). To establish
their connections in the periphery, sympathetic neuroblasts also require
several diffusible factors such as hepatocyte growth factor (HGF)
(Maina et al., 1998), artemin
(Honma et al., 2002),
neurotrophin 3 (NT3) (Francis et al.,
1999) and nerve growth factor (NGF)
(Glebova and Ginty, 2004) that
promote their survival, axon extension along blood vessels and target tissue
innervation during development.
Of the diffusible factors known to be involved in establishing sympathetic
neuron connections with peripheral target tissues, NGF has emerged as the most
important. Its role in sympathetic neuron survival and differentiation has
been known for many decades
(Levi-Montalcini and Cohen,
1960), but more recently, a particularly important role in
terminal axon extension and branching during target tissue innervation has
been identified (Glebova and Ginty,
2004). NGF may act locally to facilitate axon extension and target
tissue innervation by directly regulating neurofilament protein stabilization
(Veeranna Amin et al., 1998),
but it also alters gene expression in sympathetic neurons, which facilitates
their survival and axon outgrowth
(Milbrandt, 1987;
Riccio et al., 1997). However,
specific transcriptional regulators that control NGF-dependent gene expression
during SNS development have not been well defined.
Early growth response 1 (Egr1) protein is among a relatively small number
of transcriptional regulators induced by NGF signaling in sympathetic neurons
(Milbrandt, 1987). Disruption
of Egr-mediated gene transcription using either a dominant-negative molecule
(Levkovitz et al., 2001) or
the Egr co-repressor molecule Nab2 (Qu et
al., 1998) inhibits NGF-mediated neurite outgrowth and
differentiation in sympathetic neuron-like PC12 cells. Similarly, antisense
oligonucleotide knockdown of Egr1 protein translation inhibits neurite
outgrowth, whereas overexpression of Egr1 enhances neurite outgrowth in N2A
neuroblastoma cells (Pignatelli et al.,
1999). Thus, it is surprising that Egr1-deficient mice develop
normally and have no apparent SNS abnormalities
(Lee et al., 1995) (L.C.E. and
W.G.T., unpublished). However, considering that Egr3 and Egr4, two closely
related transcriptional regulators, have been shown to functionally cooperate
with Egr1 to regulate some target genes such as luteinizing hormone
β-peptide (LHβ), the neuroplasticity associated protein Arc and the
low-affinity neurotrophin receptor p75NTR
(Gao et al., 2007;
Li et al., 2005;
Tourtellotte et al., 2000), it
seemed plausible that other Egr proteins may have important roles in
sympathetic neuron differentiation in vivo.
Here, we identify Egr3 as having an unexpected but important role in SNS development. Egr3-deficient mice exhibit sympathetic neuron loss, target tissue innervation defects and profound dysautonomia. Egr3 expression is induced by NGF signaling and it is upregulated in sympathetic neurons during a developmental period when NGF signaling is required for normal sympathetic neuron survival and target tissue innervation in vivo. Thus, Egr3 appears to be a physiologically important effector of NGF signaling with an essential role in sympathetic neuron target tissue innervation and terminal axon branching.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Perinatal and adult (age 12-35 weeks) mice were used as indicated. Transgenic
DβH-
lacZ (DlZ) reporter mice were generated by cloning the
lacZ cDNA (Callahan and Thomas,
1994) into the BamHI site of the previously described
2949A12 DβH promoter plasmid (Hoyle
et al., 1993). F1 progeny from transgenic founder mice were
screened to establish a single reporter line with lacz expression in over
99% of sympathetic neurons and their axons (see Fig. S3 in the supplementary
material and data not shown). DlZ transgenic reporter mice were genotyped
by PCR using primers: 5'-GATTCCCCGCTAGACAAATGTGA-3' and
5'-CATGTCACCCTCCTGGTCTT-3'. All experimental procedures complied
with protocols approved by The Northwestern University Institutional Animal
Care and Use Committee.
Tissue preparation
Embryos from timed pregnant females were isolated by Cesarean section.
Postnatal mice were perfused through the heart with 0.1 M phosphate-buffered
4% paraformaldehyde (PFA, pH=7.2) and embryos were immersion fixed. Tissues
were either processed for paraffin embedding or cryoprotected overnight in
graded (15-30%) phosphate-buffered sucrose and embedded in OCT. Serial
paraffin tissue sections (16 µm) or frozen sections (12-16 µm) were
analyzed.
Ganglion neuron counts and volume estimation
SCG neuron numbers were determined using unbiased stereology and optical
dissector methods (StereoInvestigator, Microbrightfield) on every fifth serial
section from various developmental ages as previously described
(Albert et al., 2005). The
total number of neurons per SCG was estimated using an optical fractionator
probe and ganglionic volume measurements were estimated using the planimetry
function of the StereoInvestigator software.
NGF neutralization
Newborn Swiss Webster mice were injected with PBS (n=5) or
antibody (n=5; 50 mg/kg, i.p., mouse anti-NGF, clone AS-18, Exalpha
Biologicals) which has been previously well characterized for its specificity
and NGF neutralizing effects (Wild et al.,
2007). SCG were isolated 14 hours after injection and subjected to
qPCR.
Primary SCG neuron cultures
For SCG neuron cultures, E19 or P0 Swiss Webster (Charles River
Laboratories) mice or E18.5 Egr3+/+ and littermate
Egr3-/- mice were used. SCG neurons were dissociated in 1
mg/ml Type IV Collagenase (Worthington Biochemical Corporation), followed by
0.25% Trypsin-EDTA and plated on collagen-coated 35 mm dishes in Minimal
Essential Media (MEM) containing 10% fetal bovine serum (FBS), 1%
penicillin/streptomycin and 100 ng/ml of NGF. For signaling studies, neurons
were differentiated in the presence of 100 ng/ml of NGF for 7 days, deprived
of NGF for 3 hours and then re-stimulated with either vehicle (control), NGF
(100 ng/ml), NT4 (30 ng/ml) or BDNF (30 ng/ml) for 45 minutes. In some wells,
DMSO (control) or the MAP kinase kinase (MEK) inhibitor U0126 (20 µM,
Promega) were added to the cultures.
Immunohistochemistry and western blotting
Immunohistochemistry for tyrosine hydroxylase (TH; Chemicon) or
β-galactosidase (βgal; ICN Pharmaceuticals) was performed on frozen
tissue sections to identify sympathetic axons. For proliferation and apoptosis
assays, tissue sections were incubated with cleaved caspase 3 antibody (Cell
Signaling) to identify apoptotic neurons or anti-5-bromo-2-deoxyuridine (BrdU)
antibody (Sigma) to identify proliferating cells. Species appropriate
Alexa488-conjugated (Invitrogen) or Cy3-conjugated (Jackson ImmunoResearch)
secondary antibodies were used to visualize the proteins. Western blotting was
performed as described in detail (Li et
al., 2005) using the following antibodies: anti-Egr3 (sc-191),
anti-ERK1/2 (sc-94), anti-phosphorylated ERK1/2 (sc-16982) and anti-actin
(sc-1616) (all from Santa Cruz Biotechnology). The antibodies were detected
using species appropriate horseradish peroxidase-conjugated secondary
antibodies (Jackson ImmunoResearch) and SuperSignal West Pico chemiluminescent
substrate (Pierce).
lacZ enzyme histochemistry
Tissues were dissected and postfixed in 2% PFA, 0.2% glutaraldehyde, 5 mM
EGTA, 0.01% NP-40 in PBS-Mg at 4°C and reacted for 6-12 hours at 37°C
in reaction buffer (1 mg/ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium
ferricyanide). After reaction, the tissues were postfixed/dehydrated in
methanol, and cleared in 2:1 benzyl benzoate:benzyl alcohol.
In situ hybridization
In situ hybridization was performed on frozen sections using
digoxygenin-labeled antisense and sense riboprobes for Egr3 (GenBank NM018781,
nt 345-746) as described previously in detail
(Albert et al., 2005).
Apoptosis and proliferation analysis
Newborn (P0) pups received injections with 50 mg/kg, i.p. BrdU (Sigma) and
euthanized after 2 hours. BrdU and caspase 3 were detected by
immunohistochemistry and the number of immunopositive cells in every fifth
section from E15, E16, E17, E18 or P0 mice were quantified using unbiased
stereology and optical fractionator methods (StereoInvestigator,
Microbrightfield, Williston, VT). The data were reported as caspase 3+ or
BrdU+ neurons per unit volume of sampled ganglion.
qPCR
Total RNA was isolated from cultured sympathetic neurons or whole SCG using
Trizol extraction (Invitrogen). Reverse transcription and qPCR was performed
as previously described in detail (Albert
et al., 2005). The primer sequences used for expression analysis
are available upon request.
AANAT expression analysis and light cycling
Adult wild-type and Egr3-/- mice were housed in a controlled
lighting environment [10 hours of dark, 14 hours of light (10D:14L)] for at
least 3 weeks prior to analysis. ZT0 was defined as the time of dark-light
transition. Some animals were sacrificed in the dark during the dark phase of
the light-dark cycle and pineal glands were rapidly dissected. Gene expression
was determined by qPCR and compared at three time points (ZT9, ZT13 and ZT21)
between wild-type and Egr3-/- mice.
Cardiac physiology
Male and female adult wild type and Egr3-/- mice, weighing 18-23
g, were used. Pressure measurements from a 1.4 French micromanometer-tipped
Millar pressure transducer (SPR839, Millar Instruments) were calibrated
against a mercury manometer. The right jugular vein of anesthetized mice was
cannulated for fluid administration and the pressure catheter was inserted
into the right carotid artery and advanced into the left ventricle of the
heart. Heart rate and myocardial contractility (change in pressure over time;
dP/dt) measurements were compared before and after injection of the
2-adrenoreceptor antagonist, Yohimbine (YOH; 2 mg/kg, i.v.).
Heart rate and pressure data were analyzed using Millar data acquisition and
analysis software.
Statistical analysis
For SCG neuron survival assays, the data were analyzed by two-way ANOVA
using Genotype (wild type and Egr3-/-) and NGF concentration as
grouping factors. All values were expressed as mean±s.e.m. with
P<0.05 considered to be statistically significant.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), as it is often associated with abnormal sympathetic
innervation to the eyelid musculature in both humans and rodents. Because
nothing is known about Egr3 expression in sympathetic neurons, we first
examined whether it is expressed in the superior cervical ganglion (SCG),
which contains sympathetic neurons that innervate cephalic tissues including
the superior and inferior tarsal muscles of the eyelids. At embryonic day 13
(E13), when sympathetic neurons are not dependent upon NGF signaling and have
not yet innervated target tissues (Francis
and Landis, 1999; Wyatt and
Davies, 1995; Wyatt et al.,
1997), Egr3 expression was low in the SCG. Egr3 expression was
induced greater than 12-fold at E15 and greater than 22-fold by birth (P0),
two developmental time points after which sympathetic neurons are dependent
upon NGF signaling for survival and target tissue innervation
(Fig. 1A). Consistent with the
qPCR results, Egr3 expression was undetectable by in situ hybridization at E13
(Fig. 1B; see Fig. S1B'
in the supplementary material) and was markedly upregulated in most SCG
neurons by birth (P0; Fig. 1C;
see Fig. S1C' in the supplementary material). Thus, Egr3 expression is
developmentally regulated in the SCG and the timing of expression coincides
with the onset of NGF dependence and target tissue innervation. Moreover, Egr3
is regulated by NGF signaling in vivo, as neutralization of NGF function
significantly decreased Egr3 expression in SCG neurons
(Fig. 1D).
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), also
induced Egr3 expression after NGF treatment (see Fig. S2A in the supplementary
material). Also, similar to primary sympathetic neurons, NGF-mediated Egr3
expression was completely abrogated by the MEK inhibitor U0126 (see Fig. S2B
in the supplementary material). Taken together, these results indicate that
Egr3 expression is modulated by NGF signaling in sympathetic neurons,
consistent with a potential role in gene regulation related to their survival,
differentiation and/or target tissue innervation.
Sympathetic neuron loss in postnatal Egr3-deficient mice
Egr3-deficient mice have blepharoptosis similar to mice with widespread
sympathetic neuron loss in the absence of neurotrophins NGF or NT3, or their
cognate tyrosine kinase receptor TrkA
(Crowley et al., 1994;
Ernfors et al., 1994;
Farinas et al., 1994;
Smeyne et al., 1994). Unlike
neurotrophin signaling-deficient mice in which most sympathetic neuron loss
occurs during late embryonic development, no significant neuron loss was
observed in Egr3-/- mice until 1 day after birth (P1;
Fig. 2A). At P1,
Egr3-/- mice had 
30% fewer SCG neurons relative to wild type
(Fig. 2A). Moreover, consistent
with previously published observations, the total number of SCG neurons
decreased with age in both wild-type and Egr3-/- mice
(Gatzinsky et al., 2004;
Jansen et al., 2007).
Viability of senescent SCG neurons is dependent upon pro-NGF and sortilin
signaling (Jansen et al.,
2007), which does not appear to be disrupted in Egr3-/-
mice because roughly similar amounts of neuron attrition were observed in both
adult wild-type and Egr3-/- mice
(Fig. 2A).
To characterize the impact of sympathetic neuron loss on target tissue
innervation in Egr3-/- mice, transgenic reporter mice were
generated to visualize sympathetic neurons and their axons using the human
dopamine β-hydroxylase (DβH) promoter
(Hoyle et al., 1993) to
regulate expression of a -β-galactosidase fusion protein (lacZ)
(Callahan and Thomas, 1994) in
all sympathetic neurons (see Fig. S3A in the supplementary material).
Whole-mount lacZ histochemistry (see Fig. S3B-D in the supplementary
material) and double-labeling immunofluorescence for TH and β-gal (see
Fig. S3E in the supplementary material) confirmed that the neuron/axon
localized lacZ protein was a reliable and sensitive marker for all
sympathetic neurons and their axons in DβH-lacZ+ (DlZ+)
transgenic mice.
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lZ+ mice relative
to Egr3+/+:DlZ+ (wild type) littermates. The SCG
were markedly smaller in Egr3-/- mice compared with wild-type
littermates (Fig. 2B, black
arrowhead), consistent with a 30% neuron loss
(Fig. 2A) and a 36% decrease in
ganglion volume (n=3 SCG of each genotype, P<0.01). Many
axons emanating from Egr3-/- SCG were either atrophic or absent
(Fig. 2B, white arrowhead and
dashed contour, respectively). In addition, neurons that remained in the
Egr3-/- SCG were generally smaller compared with wild type (see
Fig. S4A in the supplementary material), most probably owing to atrophy, as
diameter-frequency analysis of SCG neurons showed a loss of large diameter
neurons that was accompanied by an increase in small diameter neurons (Fig.
S4B). Widespread abnormalities of the sympathetic nervous system were apparent
as the stellate (STG; Fig. 2C,
black arrowhead), thoracic (Fig.
2D, black arrowhead) and caudal paravertebral ganglia
(Fig. 2E, black arrowheads)
were all smaller in Egr3-/-: DlZ+ mice, and many
axon bundles emanating from them were also markedly thin or completely absent
compared with wild-type mice (Fig.
2C-E, white arrowheads).
Perinatal sympathetic neuron apoptosis in Egr3-/- mice
In NGF-, NT3- and TrkA-deficient mice, sympathetic neurons are generated in
normal numbers but undergo increased apoptosis during prenatal and postnatal
development (Crowley et al.,
1994; Ernfors et al.,
1994; Farinas et al.,
1994; Smeyne et al.,
1994). Sympathetic neurons were also generated in normal numbers
in Egr3-/- mice as no differences in SCG neuron number were
observed between wild-type and Egr3-/- newborn mice
(Fig. 2A). However,
significantly increased apoptosis was found in newborn Egr3-/- SCG
(Fig. 3A), which correlated
with differences in SCG neuron number that were detected slightly later at P1.
In addition, BrdU incorporation studies showed no difference in cell
proliferation between newborn wild-type and Egr3-/- SCG that could
have otherwise contributed to differences in the number of sympathetic neurons
in postnatal mice (Fig. 3B).
Thus, similar to neurotrophin- and neurotrophin signaling-deficient mice,
sympathetic neuron death in Egr3-/- mice occurs by apoptosis at a
developmental time point that coincides with active target tissue innervation
and acquisition of NGF dependence.
Embryonic and early postnatal sympathetic neurons depend upon NGF for their
survival in vitro and in vivo, and over 90% of them can be rescued from
apoptosis in vitro when 10 ng/ml of NGF is present in the culture medium
(Belliveau et al., 1997). To
examine whether Egr3-/- sympathetic neurons have an autonomous
defect in survival, wild-type and Egr3-/- SCG were isolated at
E18.5, prior to the onset of increased apoptosis that occurs in
Egr3-/- mice in vivo, and sympathetic neurons were dissociated into
culture media containing differing amounts of NGF. As expected, there was a
highly significant effect of NGF concentration on sympathetic neuron survival
for both wild-type and Egr3-/- neurons 24 hours after plating
(F3,23=123.9, P<0.0001). However, there was no
significant effect of genotype on neuron survival (F1,23=1.27,
P=0.28), indicating that Egr3-/- sympathetic neurons do
not have a cell autonomous defect in survival
(Fig. 3C).
Abnormal sympathetic target tissue innervation and terminal axon branching in Egr3-/- mice
Egr3-deficient neurons do not have an autonomous survival defect and yet
some of them die in vivo during a period of active target tissue innervation.
These observations raise the possibility that neuron death in vivo results
from a failure to normally innervate target tissues and acquire adequate
trophic factor support. To address this hypothesis, sympathetic target tissue
innervation was analyzed in adult Egr3+/+:DlZ+ and
Egr3-/-:DlZ+ mice. In all
Egr3-/-:DlZ+ target tissues examined, there was a
decrease in the overall sympathetic innervation compared with
Egr3+/+:DlZ+ target tissues, consistent with
sympathetic neuron loss. However, the remaining sympathetic axons also showed
abnormal axon extension and branching patterns within tissues. For example, in
Egr3+/+:DlZ+ submandibular and sublingual salivary
glands (Fig. 4A), which express
high levels of NGF required for normal sympathetic innervation and terminal
axon branching (Glebova and Ginty,
2004), whole-mount lacZ histochemistry highlighted robust
sympathetic axon innervation and axon branching deep into the glandular
parenchyma (Fig. 4B,
arrowheads). By contrast, in Egr3-/-:DlZ+ glands,
there was decreased innervation (Fig.
4A') that was accompanied by attenuated terminal axon
extension and branching into the glandular parenchyma
(Fig. 4B', arrowheads).
Similarly, in the trachea where there is robust sympathetic innervation to
smooth muscle and submucosal glands, the axons entered the dorsal midline and
branched to form a dense circumferential plexus in
Egr3+/+:DlZ+ mice
(Fig. 4C, white arrowheads). By
contrast, innervation to the trachea of
Egr3-/-:DlZ+ mice was generally decreased but
remaining axons also failed to branch efficiently to form a comparatively
elaborate sympathetic plexus. In some regions of the trachea the axons
appeared to barely branch at all, leaving the corresponding tracheal segments
nearly devoid of sympathetic innervation
(Fig. 4C', arrowheads).
Sympathetic innervation to several major organs, including kidneys, bowel and
spleen was also abnormal. For example, sympathetic axons in wild-type spleen
entered the organ along the splenic artery
(Fig. 4D, black arrowhead) and
then branched considerably upon entering the splenic parenchyma
(Fig. 4D, white arrowheads). By
contrast, although some axons also reached the spleen along the splenic artery
in Egr3-/-:DlZ+ mice
(Fig. 4D', black
arrowhead), they failed to branch normally and invade the splenic parenchyma
(Fig. 4D', white
arrowheads). This correlated with an overall decrease in sympathetic
innervation to the spleen, as indicated by an overall decrease in the
lacZ reaction product in Egr3-/-:DlZ+
spleens (Fig. 4D', arrow)
compared with wild type (Fig.
4D, arrow). Thus, in the absence of Egr3, there is decreased
innervation to many target tissues owing to sympathetic neuron loss and
innervation defects from residual axons that fail to normally branch and
invade target tissues.
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;
Ernfors et al., 1994;
Gurwood, 1999;
Smeyne et al., 1994). Compared
with wild type (Fig. 5A),
Egr3-/- mice have prominent blepharoptosis
(Fig. 5A'), suggesting
that sympathetic innervation to tarsal musculature may be impaired. Indeed,
compared with Egr3+/+:DlZ+ mice, which showed dense
sympathetic innervation to the superior and inferior tarsal muscles in the
eyelids (Fig. 5B, black
arrowheads) and the secretory Meibomian glands
(Fig. 5B, columnar innervation,
white arrowheads), innervation to the same structures in
Egr3-/-:DlZ+ mice was nearly absent
(Fig. 5B'). Also similar
to humans with familial dysautonomia, the blepharoptosis in adult
Egr3-/- mice was accompanied by corneal ulcerations, which were
most likely the result of a chronic lack of corneal lubrication from Meibomian
glands, which require sympathetic innervation for normal secretomotor function
(Fig. 5C', arrow).
The pineal gland, an SCG target tissue that produces the hormone melatonin,
is involved in synchronizing circadian rhythms with environmental light cues.
During the dark phase of the light-dark cycle, arylalkylamine N-acetyl
transferase (AANAT), the rate-limiting enzyme for melatonin synthesis, is
transcriptionally upregulated in pinealocytes by increased sympathetic
activity (Borjigin et al.,
1995; Foulkes et al.,
1997). Wild-type (Fig.
6A) and Egr3-/-
(Fig. 6A') pineal glands
were morphologically indistinguishable. Sympathetic innervation detected by TH
immunohistochemistry showed that wild-type pineal glands were innervated by a
dense network of TH+ axon terminals
(Fig. 6B), whereas pineal
glands from Egr3-/- mice had almost none
(Fig. 6B'). Similarly, a
dense plexus of lacZ+ (sympathetic) axons was observed in
whole-mount wild-type pineal glands (Fig.
6C) which was markedly diminished in Egr3-/- pineal
glands from DlZ+ mice (Fig.
6C'). To examine whether sympathetic denervation was also
associated with deregulation of AANAT expression during the light-dark cycle,
wild-type and Egr3-/- mice were entrained to a 10D:14L light-dark
cycle (see Materials and methods). Pineal glands were dissected for qPCR
analysis at three time points during the 24-hour photo period: ZT13, ZT21 and
ZT9, with ZT0 defined as the temporal onset of the dark-light transition.
Wild-type pineal glands showed a characteristic pattern of AANAT expression
associated with the entrained light-dark cycle. Very low basal AANAT
expression was observed near the end of the light cycle (ZT13), it was highly
induced during the dark phase when sympathetic activity to the pineal gland is
maximal (ZT21, defined as 100% maximal induction in this paradigm) and it
rapidly declined to basal levels after the onset of the entrained light phase
(ZT9; Fig. 6D). By contrast, in
Egr3-/- pineal glands, AANAT induction reached less than 20% of the
maximal wild-type induction (ZT21) after the onset of the dark phase before
returning to baseline during the light phase of the cycle
(Fig. 6D). These results are
consistent with decreased sympathetic innervation to the pineal gland, which
leads to impaired AANAT induction during the dark phase of the light cycle in
Egr3-/- mice. Moreover, the results raise the possibility that
Egr3-/- mice have altered circadian rhythms as a consequence of
sympathetic dysautonomia and impaired AANAT cycling and melatonin
synthesis.
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). To examine the impact of sympathetic neuron loss on cardiac
sympathetic innervation, we compared the pattern of sympathetic innervation in
hearts from adult wild-type and Egr3-/- mice. In hearts from
Egr3+/+:DlZ+ mice, prominent epicardial innervation
to the right atrium (Fig. 7A)
and ventricles (Fig. 7B,C) was
observed. The ventricular epicardial axons formed highly branched networks of
small axons that diffusely innervated the myocardium
(Fig. 7C, arrowhead). In
Egr3-/-:DlZ+ hearts, there was a marked decrease in
the innervation to the inferior surface of the right atrium and to both
ventricles (Fig.
7A'-C'), consistent with sympathetic neuron loss.
Similar to other sympathetic target tissues that we examined, there was a
conspicuous absence of terminal axon branching on the surface of the
ventricles (compare Fig. 7C with
7C'). To determine whether these abnormalities have
physiological significance, heart rate and myocardial contractility were
measured in cardiac catheterized adult wild-type and Egr3-/- mice.
The catheterized mice were administered the central
2-adrenergic receptor antagonist yohimbine (YOH) to increase
post-ganglionic sympathetic activity. Whereas the baseline heart rate and
myocardial contractility of untreated wild-type and Egr3-/- mice
were similar, YOH treatment of wild-type mice increased heart rate and
contractility greater than twofold relative to baseline. By contrast, YOH
treatment of Egr3-/- mice resulted in a significantly blunted
increase in heart rate (Fig.
7D) and contractility (Fig.
7E). Thus, cardiac hemodynamic abnormalities are present in
Egr3-/- mice as a consequence of impaired sympathetic innervation
to the heart. Taken together, these results demonstrate that
Egr3-/- mice have profound sympathetic dysautonomia associated with
the structural SNS abnormalities. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Egr1 was
later shown to have an important role in NGF-mediated neurite outgrowth in
PC12 and N2A neuroblastoma cells, but a role in SNS development was not
confirmed in Egr1-deficient mice (Lee et
al., 1995). Despite the fact that Egr3 is co-expressed with Egr1
in many cell types and they can cooperate to directly regulate some target
genes (Carter and Tourtellotte,
2007; Gao et al.,
2007; Li et al.,
2005), it is surprising to find that Egr3, but not Egr1, has an
essential role in SNS development in vivo.
We found that Egr3 expression was upregulated in post-migratory SCG neurons
at a time point that coincides with their dependence on NGF for survival and
target tissue innervation. This correlation was further substantiated by in
vitro and in vivo experiments which showed that Egr3 is regulated in primary
and transformed sympathetic neurons by NGF signaling. Moreover, NGF-mediated
Egr3 expression was entirely dependent upon MEK (Erk1/2) signaling, which is
well known to enhance NGF-dependent axon outgrowth in sympathetic neurons
(Atwal et al., 2000;
Lein et al., 2007;
Thompson et al., 2004).
Although these results do not rule out non-neurotrophin-mediated mechanisms of
regulation or a non-neuron autonomous function for Egr3, they clearly
demonstrate that Egr3 expression is modulated by NGF signaling and that it is
expressed by sympathetic neurons at a time when it could influence gene
expression involved in their differentiation and target tissue
innervation.
A role for Egr3 in SNS development was confirmed in Egr3-deficient mice.
Sympathetic neuron loss was first detected in SCG neurons 1 day after birth,
which was preceded by a significant increase in apoptosis. Although the
quantitative and in vitro studies were focused on SCG neurons, a detailed
analysis of Egr3-/- mice demonstrated widespread atrophy of
sympathetic chain ganglia and target tissue innervation defects, indicating a
global role for Egr3 in SNS development. However, unlike mice lacking NGF or
its receptor TrkA, which lose most sympathetic neurons by birth
(Crowley et al., 1994;
Smeyne et al., 1994), we found
that only about one-third of SCG neurons were lost in Egr3-deficient mice
after birth, despite the fact that it is expressed in most, if not all, SCG
neurons. This discrepancy between Egr3 expression and neuron survival may be
explained by the fact that Egr3 does not have a neuron-autonomous role in
survival as there was no difference in survival between wild-type and
Egr3-/- neurons when NGF was supplied in culture media in vitro.
Yet, sympathetic neuron death is observed in vivo, which results in many
relatively hypoinnervated target tissues that also have residual innervation
with abnormal terminal axon extension and branching. Thus, partial loss of
sympathetic neurons in vivo is most probably a consequence of inadequate
trophic factor acquisition because of abnormal, but not completely absent,
target tissue innervation in Egr3-/- mice
(Korsching and Thoenen, 1983).
Similarly, sympathetic neuron death which was detected at P0 in Egr3-deficient
mice was delayed compared with TrkA-deficient mice where neuron death begins 2
days earlier at E17.5 (Fagan et al.,
1996). Sympathetic neuron death may occur later in Egr3-deficient
mice because abnormal terminal axon extension and branching within target
tissues, which occurs relatively late during SNS development, would be
expected to lead to relatively delayed defects in NGF acquisition.
Egr3, a potential transcriptional effector of NGF signaling
The density of sympathetic innervation is correlated with the timing and
extent of NGF production by target tissues
(Korsching and Thoenen, 1983;
Korsching and Thoenen, 1988;
Shelton and Reichardt, 1984).
Sympathetic neurons depend upon NGF for their survival
(Crowley et al., 1994;
Levi-Montalcini and Booker,
1960) and for terminal axon extension and branching within target
tissues (Glebova and Ginty,
2004; Kuruvilla et al.,
2004). Although Egr3 does not appear to have a direct role in
sympathetic neuron survival like NGF, it does appear to have a similar role in
sympathetic terminal axon extension and branching within target tissues. In
the absence of Egr3, we observed a heterogeneous effect on sympathetic target
tissue innervation with some tissues showing highly diminished innervation
(e.g. eyelids and pineal gland), while in other tissues there was moderate
loss of innervation associated with prominent terminal axon extension and
branching defects (e.g. salivary glands, trachea, spleen and heart). These
results are strikingly similar to NGF/Bax double knockout (dKO) mice, where
inhibition of NGF- and Bax-dependent sympathetic neuron death
(Deckwerth et al., 1996) made
it possible to study target tissue innervation in the absence of NGF
(Glebova and Ginty, 2004).
Although NGF/Bax dKO mice died shortly after birth before the sympathetic
nervous system was completely developed, sympathetic axons extended along
vessels and encroached upon target tissues where they either did not innervate
them or they innervated them with highly attenuated terminal axon extension
and branching. Thus, although Egr3 is just one of many genes regulated by NGF
signaling within sympathetic neurons, it is reasonable to assume that it has a
particularly important role in NGF-mediated gene expression involved in target
tissue innervation. To better understand how Egr3 mediates particular aspects
of NGF signaling, it will be important for future studies to identify and
characterize the target genes regulated by Egr3 in sympathetic neurons.
Sympathetic dysautonomia in Egr3-deficient mice
Unlike mice lacking NGF or TrkA, most Egr3-deficient mice survive past the
perinatal period with many signs and symptoms of sympathetic dysautonomia
because of abnormal, but not completely absent, sympathetic target tissue
innervation. In Egr3-/- mice, blepharoptosis was a result of
impaired sympathetic innervation to the tarsal musculature of the eyelid and
corneal ulcerations resulted from abnormal secretomotor function of tarsal
Meibomian glands, which normally provide lubrication to the surface of the
eye. In addition, denervation of the pineal gland was found to disrupt light
cycle-dependent and sympathetic activity-induced AANAT expression, which is
required for normal melatonin synthesis and circadian rhythms. Cardiac
function was also impaired by poor sympathetic terminal axon extension and
branching to the myocardium, which resulted in abnormal regulation of heart
rate and contractility.
Egr3 has not been previously implicated in SNS development or human dysautonomia but it is remarkable that Egr3-deficient mice have physiological abnormalities similar to humans with dysautonomia. The etiology of most human dysautonomias is unknown, but some congenital cases have been associated with abnormal NGF signaling and transcriptional regulation. For example, hereditary sensory and autonomic neuropathy (HSAN) type IV is associated with mutations in the TrkA gene (OMIM #256800), while HSAN type V is associated with mutations in the NGFβ gene (OMIM #608654). Similarly, HSAN type III (Riley-Day syndrome; familial dysautonomia), the most common congenital dysautonomia, is associated with mutations in the IKBKAP gene, which encodes a protein component of the halo-transcriptional elongator complex (OMIM #223900). Taken together with this new evidence that Egr3 has an important role in SNS development and NGF signaling, it seems plausible that Egr3-dependent gene regulation could be involved in some forms of congenital and/or acquired human dysautonomia.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/17/2949/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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